Macrophage miRNAs in atherosclerosis

BBAMCB-57901; No. of pages: 7; 4C: 2 Biochimica et Biophysica Acta xxx (2016) xxx–xxx

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Macrophage miRNAs in atherosclerosis☆ Denuja Karunakaran a, Katey J. Rayner a,b,⁎ a b

University of Ottawa Heart Institute, Ottawa, Canada Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Canada

a r t i c l e

i n f o

Article history: Received 20 December 2015 Received in revised form 6 February 2016 Accepted 6 February 2016 Available online xxxx Keywords: MicroRNA Macrophage Cholesterol efflux Atherosclerosis Inflammation Therapeutic

a b s t r a c t The discovery of endogenous microRNAs (miRNAs) in the early 1990s has been followed by the identification of hundreds of miRNAs and their roles in regulating various biological processes, including proliferation, apoptosis, lipid metabolism, glucose homeostasis and viral infection Esteller (2011), Ameres and Zamore (2013) [1,2]. miRNAs are small (~ 22 nucleotides) non-coding RNAs that function as “rheostats” to simultaneously tweak the expression of multiple genes within a genetic network, resulting in dramatic functional modulation of biological processes. Although the last decade has brought the identification of miRNAs, their targets and function(s) in health and disease, there remains much to be deciphered from the human genome and its complexities in mechanistic regulation of entire genetic networks. These discoveries have opened the door to new and exciting avenues for therapeutic interventions to treat various pathological diseases, including cardiometabolic diseases such as atherosclerosis, diabetes and obesity. In a complex multi-factorial disease like atherosclerosis, many miRNAs have been shown to contribute to disease progression and may offer novel targets for future therapy. This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernández-Hernando and Yajaira Suárez. © 2016 Elsevier B.V. All rights reserved.

1. miR biogenesis miRNAs are non-coding RNAs embedded within the intergenic or intronic regions of host genes and are usually transcribed by RNA polymerase II as a primary miRNA (pri-miRNA) often concurrently with their host genes (Fig. 1) [1–4]. After transcription, the pri-miRNA is cleaved by the microprocessor complex consisting of the nuclease Drosha and the DiGeorge syndrome critical region in gene 8 (DGCR8) in mammals to liberate the precursor-miR (pre-miRNA). The premiRNAs are exported through nuclear pore complexes by exportin 5 and Ran-GTP into the cytoplasm, where they undergo additional processing. The initial cleavage by Drosha in the nucleus defines one end of the mature miRNA, while the other end of pre-miRNA is digested in the cytoplasm after nuclear export by the endonuclease RNAse III Dicer to generate the mature miRNA duplex, consisting of the miRNA (guide) and miRNA* (passenger) strands. These individual strands, which are often denoted as -5p or -3p, are then incorporated into an Argonaute family protein complex to generate the RNA-induced Abbreviations: miRNA, microRNA; anti-miR, anti-microRNA; apo, apolipoprotein; SREBP, sterol response element binding protein; HDL, high density lipoprotein; LDL, low density lipoprotein; VLDL, very low density lipoprotein; ABC, ATP-binding cassette transporter; NPC1, Niemann–Pick C1; HFD, high fat diet. ☆ This article is part of a Special Issue entitled: MicroRNAs and lipid/energy metabolism and related diseases edited by Carlos Fernández-Hernando and Yajaira Suárez. ⁎ Corresponding author at: Department of Biochemistry, Microbiology and Immunology, Faculty of Medicine, University of Ottawa, Canada. E-mail address: [email protected] (K.J. Rayner).

silencing complex (RISC). The RISC binds sites with partial complementarity between the seed region of the miRNA and target sequences on the 3′ untranslated region (UTR) of mammalian messenger RNA (mRNA). This binding induces target mRNA degradation by deadenylation and endonucleolytical cleavage or translational repression by promoting ribosome drop-off or stimulating proteolysis of the nascent peptides [3,4]. Until recently, it was thought that miR* strands did not have a function and were generally degraded but recent evidence suggest that these strands too have specific mRNA targets [5]. 2. Reverse cholesterol transport (RCT) and atherosclerosis Atherosclerosis is a maladaptive, multifactorial disease initiated by modified low-density lipoprotein (LDL) trapped within the vessel wall and maintained by a chronic inflammatory response [6]. Monocytes are recruited into the intima, where they differentiate into macrophages that avidly take up the modified LDL to become macrophage foam cells. These foam cells secrete a wide array of cytokines and chemokines that promote the recruitment of additional monocytes and promote the proliferation of macrophages to further propagate lesion expansion. Under homeostatic conditions, any excess intracellular cholesterol within the macrophage foam cell is released onto nascent high-density lipoprotein (HDL) particles by the interaction of ATP-binding cassette A1 (ABCA1) transporters on the macrophage membrane and circulating poorly lipidated apolipoprotein (apo) A-I lipoproteins. These lipid-loaded HDL particles, in turn, return to the liver for cholesteryl ester uptake

http://dx.doi.org/10.1016/j.bbalip.2016.02.006 1388-1981/© 2016 Elsevier B.V. All rights reserved.

Please cite this article as: D. Karunakaran, K.J. Rayner, Macrophage miRNAs in atherosclerosis, Biochim. Biophys. Acta (2016), http://dx.doi.org/ 10.1016/j.bbalip.2016.02.006

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Fig. 1. miRNA regulation of macrophage function in atherosclerosis. miRNAs are transcribed in the nucleus by RNA polymerase II (Pol II) as primary miRNA, which is then cleaved by Drosha and the DiGeorge syndrome critical region in gene 8 (DGCR8) and exported into the cytoplasm as a precursor miRNA. It is further processed by the RNA-induced silencing complex (RISC). Mature miRNA binds to its target mRNA to either induce its degradation or repress its translation. A number of miRNAs regulate cholesterol efflux by targeting ABCA1, including miR-33, miR-26, miR-27a, miR-758, miR-128-1, miR-148a, miR-29, miR-144, miR-302a, miR-378. miR-33 also targets genes involved in mitochondrial function (AMPKα, PGC-1α), fatty acid oxidation (CPT1α, CROT, HADHB) and macrophage polarization (AMPKα). Activation of inflammatory TLRs activates miR-155, which is thought to promote macrophage inflammation. In contrast, apoE can promote miR-146a expression in macrophages to inhibit TLR-activated IRAK1 and TRAF6 to reduce macrophage inflammation. miRNAs in red have been studied in vivo, whereas miRNAs in black have been studied in vitro. miRNAs in pink are upregulated during either pro-inflammatory M1 or anti-inflammatory M2 polarization.

to be excreted into the feces — a process called reverse cholesterol transport (RCT) [7]. Following efflux, macrophages are lipid-depleted, which allows them to emigrate out of the intima to reduce lesional inflammation. However, in the setting of excess circulating cholesterol and its accumulation within the intima, this emigration process is impaired and together with defective efferocytosis, leads to lesion expansion [6]. While HDL is a critical component of the RCT process, and has been found to be controlled by a number of miRNAs, including miR-33, miR-148a and miR-144 [8–14], these HDL-regulatory miRNAs have been reviewed extensively elsewhere [15–19]. Thus, we will focus our discussion specifically on miRNAs that have been shown to alter cholesterol efflux capacity in macrophages (Fig. 1). 3. miRNA control of cholesterol efflux and ABC transporters Maintenance of intracellular cholesterol homeostasis is vital for cell survival and function and involves many layers of tightly regulated processes. Cholesterol depletion induces the activation of transcription factors sterol regulatory element-binding transcription factor (SREBF) 1 and 2, which encode the 3 mammalian sterol regulatory elementbinding protein (SREBP) isoforms: SREBP-1a, SREBP-1c and SREBP-2 which have diverse yet interacting roles in lipogenic transcriptional regulation [20]. The SREBP family in turn triggers genes that regulate fatty acid synthesis (fatty acid synthase; FASN), cholesterol synthesis (3-hydroxy-3-methylglutaryl-CoA reductase; HMGCR) and cholesterol uptake (low density lipoprotein receptor; LDLR) that restore intracellular lipid balance [21]. In contrast, when lipids are in excess, efflux

pathways are engaged to rid the cell of toxic accumulation of cholesterol. Metabolism of intracellular cholesterol produces oxysterol intermediates that activate the liver X receptor alpha (LXRα) transcription factors to induce the expression of the cholesterol transporters ABCA1 and ABCG1. These transporters remove excess cholesterol and phospholipids by interaction with lipid acceptors apoA-I, apoE and mature HDL particles. In addition, excess lipid promotes the autophagic machinery to remodel the lipid droplet to mobilize free cholesterol to ABCA1 for extracellular release [22]. Perhaps not surprisingly, the balance of intracellular cholesterol is a tightly regulated and highly conserved process, and is controlled extensively through miRNAs. One of the most well characterized miRNAs regulating cholesterol efflux is miR-33a/b (miR-33a in mice) embedded in the intronic regions of human and mouse Srebf-2 and its sister miR-33b, embedded within human Srebf-1 [12,14,23]. During cholesterol depletion, miR-33 is co-transcribed with its host gene to restore sterol balance by negatively regulating lipid export. The strongest known miR33 gene target is the cholesterol transporter, ABCA1, which contains multiple miR-33 binding sites highly conserved across humans and large mammals and its suppression by miR-33 acts to inhibit cholesterol efflux [24]. miR-33 is also known to target other cholesterol transporters, ABCG1 (in mice only) and Niemann–Pick C (NPC1, humans only), which acts to further limit cholesterol efflux during times of sterol depletion [23]. miR-33a and miR-33b differ by only 2 nucleotides outside the seed region responsible for target detection and thus, are predicted to target similar subset of genes [24]. Given its ability to block cholesterol efflux [12–14,23], it was surmised that inhibiting miR-33

Please cite this article as: D. Karunakaran, K.J. Rayner, Macrophage miRNAs in atherosclerosis, Biochim. Biophys. Acta (2016), http://dx.doi.org/ 10.1016/j.bbalip.2016.02.006

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in macrophages would de-repress ABCA1 expression and promote cholesterol efflux which in turn would limit atherosclerosis. In the majority of studies, anti-miR33 therapy using anti-sense oligonucleotides or genetic deletion consistently promoted cholesterol efflux from macrophages to ultimately reduce plaque progression or promote its regression in atherosclerotic mouse models [11,25–30]. Notably, 2′fluoro/ methoxyethyl (2′F/MOE) modified anti-miRNAs directly penetrated macrophages within the vessel wall, arguing that anti-miR33 therapy could serve to directly promote cholesterol efflux in foam cells [26]. In humans, we recently reported that miR-33a and miR-33b expression is markedly increased in carotid atherosclerotic plaques relative to control arteries [29] and hypercholesterolemic patients have a marked increase in miR-33b expression in carotid plaques compared to normocholesterolemic patients [31] supporting the idea that therapeutic miR-33 inhibition could reduce atherosclerotic lesions in humans in a similar manner to what was observed in experimental mouse models. Besides targeting cholesterol transporters, miR-33 has been found to block the expression of a number of genes involved in energy balance in the cell. Our group recently identified novel mitochondrial miR-33 targets in macrophages, including peroxisome proliferatoractivated receptor-γ coactivator (PGC)-1α and pyruvate dehydrogenase lipoamide kinase isozyme 4 (PDK4), which promote mitochondrial biogenesis, respiration and ATP production to mediate macrophage cholesterol efflux [29]. In macrophages transfected with miR-33 inhibitors, PGC-1α expression was markedly increased, which in turn increased cellular respiration, mitochondrial biogenesis and ATP production [29]. In mice with type 1 diabetes, anti-miR33 treatment accelerated the regression of atherosclerosis, total macrophage content in the plaque and inflammatory gene expression [32], suggesting that regulation of macrophage energy balance and inflammatory phenotype can ameliorate metabolic dysfunction. Interestingly, a recent study demonstrated that anti-miR33 therapy or genetic knockout of miR-33 in neural cells promotes cholesterol efflux by targeting ABCA1 and increases apoE lipidation, resulting in decreased endogenous amyloid-β protein in the cortex in a mouse model of Alzheimer's disease [33]. Whether miR-33 regulates apoE lipidation in macrophages is yet to be determined, but given the importance of macrophage-specific apoE in promoting cholesterol efflux and reducing atherosclerosis [34], it is anticipated that anti-miR33 therapy targeting ABCA1 would also affect macrophage apoE. Together, these data highlight how one specific miR (e.g. miR-33) can target multiple genes across various genetic networks (e.g. cholesterol transporters, mitochondrial genes, HDL biogenesis) to coordinately regulate cholesterol efflux. In addition to miR-33, other miRNAs have been shown to regulate ABCA1 expression in macrophages. miR-144 is an intergenic miR present in a bicistronic cluster with miR-451 and its expression is regulated by both Farnesoid X Receptor (FXR) and Liver X Receptor (LXR) — two nuclear hormone regulators of cholesterol metabolism. Similar to miR-33, miR-144 was found to directly target the 3′UTR of ABCA1 in mouse and human hepatocytes to dampen cholesterol efflux to apoA-I. In vivo, antagonism of miR-144 raised hepatic expression of ABCA1 and circulating HDL [8,9]. In these initial studies, macrophage cholesterol efflux was examined in vitro upon miR-144 inhibition, but atherosclerosis progression in vivo was not assessed. In contrast, over-expression of miR-144 was found to accelerate atherosclerosis by targeting ABCA1 in the vessel wall and reducing RCT in vivo [35]. Therefore, it follows that inhibiting miR-144 would protect against atherosclerosis by raising macrophage and hepatic ABCA1 expression, but this has yet to be shown in vivo. Other miRNAs that repress ABCA1 expression and cholesterol efflux include miR-26, miR-27a, miR-758, miR-128-1 and miR-148a [10,36–39]. Like miR-33, overexpression of miR-26, miR-27a and miR-758 in macrophages reduces cellular ABCA1 and cholesterol efflux [36–38]. Of note, miR-758 has also been shown to be elevated in carotid plaques of hypercholesterolemic patients, suggesting that it may indeed target macrophage cholesterol

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efflux in vivo [31]. While each of these miRNAs shows promise in regulating cholesterol efflux and RCT, many have yet to be tested in vivo to evaluate their true therapeutic potential in improving macrophage cholesterol efflux capacity and atherosclerotic progression. Recently, miR-302a was identified in a genome-wide screen of cholesterol-responsive miRNAs as being one of the strongest miRNAs modified by intracellular cholesterol [40]. After loading macrophages with acetylated LDL, 47 miRNAs were differentially expressed, with miR-302a showing the most significant downregulation in response to cholesterol loading. miR-302 directly targets the 3′UTR of ABCA1 and alters cholesterol efflux in mouse macrophages. In vivo inhibition of miR302a upregulated liver and aorta expression of ABCA1, raised circulating HDL by 35%, and reduced atherosclerotic lesion area in Ldlr−/− mice fed an atherogenic diet [40]. Although miR-302a has also been shown to negatively regulate peroxisome proliferator-activated receptor gamma (PPARγ) in adipocytes, the other targets of miR-302a in atherosclerosis remain to be elucidated [41].

4. Macrophage inflammatory miRNAs As discussed above, accumulation of LDL within the vessel wall results in its modification via oxidation and aggregation, which in turn, acts a stimulus for the innate immune system [6]. Modified LDL can either (i) be phagocytosed to induce intracellular cholesterol accumulation and NLRP3 inflammasome activation and/or (ii) bind directly to macrophage pattern recognition receptors such as Toll-like receptors (TLRs) to activate pro-inflammatory signaling pathways [6,42,43]. Activation of TLR signaling stimulates cytokine and chemokine production that propagates the inflammation that expands the atherosclerotic lesions. One of the most well-investigated and controversial miRs in atherosclerotic inflammation is miR-155, which is encoded in the B cell integration cluster (Bic) gene [44]. Mildly oxidized LDL and interferon γ (IFNγ) or activation of TLRs induces the expression of miR-155, which in turn, represses suppressor of cytokine signaling 1 (SOCS-1) and B-cell lymphoma 6 (Bcl-6) and promotes pro-inflammatory cytokines CCL2, IL-5, NOS2 and TNFα to mediate macrophage inflammation [45–49]. Of note, miR-155 is highly expressed in both mouse and human aortic atherosclerotic lesions and as such, has been touted as a potential therapeutic target to treat advanced atherosclerosis [46,49, 50]. However, to date, the role of miR-155 in atherosclerosis is under debate. Transplant of miR-155-deficient bone marrow cells into Ldlr−/− mice fed a western diet amplifies macrophage inflammation, increases atherosclerotic lesions and decreases plaque stability, suggesting that miR-155 is atheroprotective [51]. In contrast, bone marrow transplant of miR-155−/− cells into Apoe−/− mice fed a western diet decreases lesional macrophages and reduces atherosclerotic lesion size, implying miR-155 is pro-atherogenic [46]. Consistent with the latter study, whole body knockout of miR-155 in Apoe−/− mice fed a high cholesterol diet also showed markedly reduced macrophage inflammation, cholesterol efflux and decreased atherosclerotic lesions in the aortic root [47]. Importantly, the differences in the ability of macrophage miR-155 to reduce lesion progression may greatly depend on the stage of atherosclerotic development (i.e. early versus advanced) when the expression of key target genes is stage-specific [52]. Nonetheless, these findings highlight how miRNA-based control of dynamic cellular processes needs to be carefully and thoroughly examined before the potential of miRNA therapy is considered. The controversy surrounding miR-155 also extends to other models of vascular disease. For example, although miR-155 demonstrated antiangiogenic properties in vitro, femoral artery ligation in miR-155−/− mice markedly obscured revascularization and arteriogenesis due to defective monocyte infiltration into the damaged area [53]. Thus, this study showed that the pro-arteriogenic properties of miR-155 offset the anti-angiogenic properties of miR-155 [54]. This study highlights how caution must be taken in designing therapies that globally target

Please cite this article as: D. Karunakaran, K.J. Rayner, Macrophage miRNAs in atherosclerosis, Biochim. Biophys. Acta (2016), http://dx.doi.org/ 10.1016/j.bbalip.2016.02.006

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miR-155 as the simultaneous targeting of multiple genes in different cell types could present as a double-edged sword. A related miRNA, miR-342-5p, also regulates inflammation in part by regulating the expression of miR-155. Activation of miR-342-5p expression by the transcription factor PU.1 during macrophage differentiation directly promotes RAC-alpha serine/threonine-protein kinase (Akt1) to induce M2 macrophage phenotype (see below) and represses miR-155 and nitric oxide synthase 2 (Nos2) to inhibit pro-inflammatory cascades [55]. Thus, systemic inhibition of miR-342-5p decreases atherosclerosis and presents as another novel therapeutic target. However, due to the controversy related to miR-155, similar considerations must be taken when targeting miR-342-5p. Cell-specific or disease-stage specific targeting of these miRNAs could still have promising therapeutic outcomes and further investigations are urgently needed to address these issues. For decades, it has been understood that apoE, and in particular macrophage-specific apoE, is atheroprotective [56–58]. Mice deficient in Apoe spontaneously develop atherosclerosis, which can be accelerated upon feeding a western diet, and Apoe−/− mice are now an established model of atherosclerosis. Evidence continues to grow showing that apoE not only promotes cholesterol efflux, lipoprotein metabolism (e.g. VLDL lipolysis) and clearance but also has antiinflammatory properties that contribute to the reduction of atherosclerosis [34,59–61]. Recently, Li et al. [62] have identified that apoE directly upregulates PU.1-dependent macrophage miR-146a expression, which in turn represses nuclear factor-κB (NF-κB) signaling cascades via direct inhibition of miR-146a target genes Interleukin-1 receptor-associated kinase (IRAK) 1 and TNF receptor-associated factor (TRAF) 6. These act as negative regulators of the NF-κB inhibitor IKKB, and thus miR146 overexpression leads to down-regulation of NF-κB activation and in turn reduces atherosclerosis [62]. It remains to be determined whether apoE simultaneously targets other miRNAs that regulate inflammatory cascades in macrophages and whether apoE mediated regulation of miR-146a also affects other known miR-146a targets such as IRAK2, myeloid differentiation primary response gene 88 (MyD88) and TLR4 [63]. Of note, deletion of miR-146a in models of inflammation reduces NF-κB activation and the induction of pro-inflammatory adhesion molecules in endothelial cells in culture, and miR-146a−/− mice are resistant to endothelial activation following treatment with IL-1β [64]. Although miR-146a/b expression is significantly increased in human atherosclerotic plaques [50], the outcome of miR-146a deletion on the development of atherosclerosis has yet to be investigated. 5. miRNAs and macrophage subsets Macrophages can be divided into different subsets according to their capacity to mount either a pro- or anti-inflammatory response, along with the expression of specific cell markers. A wide spectrum of macrophage types have been described in vivo in atherosclerotic lesions: proinflammatory [M1, M4], anti-inflammatory [M(Hb), Mhem, M2a, M2b, M2c] and anti-oxidant [Mox] (reviewed in [65]). However, in vitro macrophages have been divided into 2 key classes: pro-inflammatory M1 macrophages and anti-inflammatory M2 macrophages. The M1 macrophage subset expresses NOS IL-1β, TNFα, IL-6, IL-12, IL-23, CXCL9, CXCL10 and CXCL11 and secretes pro-inflammatory cytokines (e.g. TNFα, IL-6, IL-12) in response to injury or infection [65,66]. M1 macrophages have been shown to promote the progression of atherosclerosis, and are abundant within lesions of progressing plaques [67,68]. Conversely, macrophages that resolve inflammation by inciting repair pathways are termed M2 macrophages, and express LXRα, CD163, arginase I, IL-4, IL-10 and IL-13 [65]. These M2 macrophages are characteristic of regressing atherosclerotic plaques, and are thought to be required for the resolution of inflammation within the lesion (reviewed by [66]). Naturally, miRNAs have been found to be differentially regulated in different macrophage subsets. Human macrophages treated with lipopolysaccharide (LPS) and INFγ to induce pro-inflammatory M1

macrophages have increased expression of miR-29b-1, miR-125a, miR-26a-2*, miR-155 and miR-155*, whereas anti-inflammatory macrophages induced by alternative activation (M2a) or “type 2” activation (M2b), have increased expression of miR-193b or miR-27a*, miR-29b-1*, miR-132*, miR-222* and miR-155* respectively [45,46, 69]. In vitro overexpression of miR-155 and miR-29b induced M1 phenotypic expression of IL-6, TNFα and CXCL9, confirming that the upregulation of these miRNAs is pro-inflammatory [69]. Conversely, anti-inflammatory therapy with coenzyme Q10 enhances miR-378 which in turn increases ABCG1 expression and increased reverse cholesterol transport and reduced atherosclerosis [70]. Consistent with differential miRNA expression upon various inflammatory stimuli, two independent studies have shown that in vitro treatment of THP-1 macrophages with pro-inflammatory cytokine(s), IL-6 or both IL-6 and TNFα, have increased expression of miR-106 or miR-33a respectively, both of which repress ABCA1 expression and promote cholesterol retention [71,72]. Similarly, pathogenic infection of macrophages in vitro with Chlamydia pneumoniae also represses ABCA1 and cholesterol efflux by upregulating the expression of both miR-33 and TLR2 [73]. In addition to regulating key cholesterol efflux and mitochondrial genes, miR-33 has recently been shown to directly repress 5′ adenosine monophosphate-activated protein kinase (AMPKα) to regulate genes that affect macrophage polarization [25]. Blocking miR-33 in macrophages in vitro and in vivo de-represses AMPKα to decrease glycolysis, increase fatty acid oxidation and enhance retinal dehydrogenase [aldehyde dehydrogenase 1 family, member A2 (ALDH1A2)/ retinaldehyde dehydrogenase (RALDH)], which in turn boosts FOXP3+ Tregs to promote M2 macrophage phenotype [25]. Together, these studies highlight the complex regulation of miRNAs such as miR-33 and their various target genes and downstream effectors that are simultaneously regulated. They also demonstrate the limitations in our knowledge of miRNA function and targets, and the importance of further exploring novel pathways that are concurrently targeted by specific anti-miRNA therapies. 6. Targeting the plaque macrophage: the promise of miRNA therapeutics Given the importance of HDL in the removal of cholesterol from the vessel wall, HDL raising therapies became highly sought-after as a treatment to reduce atherosclerosis burden in the setting of optimal LDL lowering with statins [7]. However, recent genetic studies showing lack of association between genetic variants in HDL and cardiovascular risk together with the failure of large-scale clinical trials of cholesterylester transfer protein (CETP) inhibitors to lower cardiovascular events have called this paradigm into question [74,75]. Conversely, it is now understood that the capacity for macrophages to efflux cholesterol onto nascent HDL particles into the RCT pathway is the key factor in determining HDL functionality [76,77]. With the growing understanding of how miRNAs control cholesterol efflux, these could offer a novel therapeutic opportunity to regress atherosclerosis that specifically target plaque macrophages to reduce cholesterol in the vessel wall. The pharmacological inhibition of miRNAs to treat human diseases is continuing to show promise, and in the past few years, there has been a surge in the development of RNA-based therapeutics in both preclinical and clinical trials. Almost simultaneously with the discovery of miRNAs, the first generation of anti-sense oligonucleotide miRNA inhibitors was developed and delivered to non-human primates and showed effective inhibition of the target miR-122 [78]. Anti-miRNAs are single stranded and are formulated with chemically modified backbones that render the oligonucleotides resistant to nuclease degradation, allowing anti-miRNAs to be administered easily and safely via intravenous or local delivery [79,80]. Newer chemistries have been developed to modify anti-miRNAs to improve cellular uptake and affinity for the intended miRNA target, reducing the effective dose required for in vivo miRNA

Please cite this article as: D. Karunakaran, K.J. Rayner, Macrophage miRNAs in atherosclerosis, Biochim. Biophys. Acta (2016), http://dx.doi.org/ 10.1016/j.bbalip.2016.02.006

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inhibition. Due to its promising pre-clinical efficacy, miR-122 was one of the first miRNA inhibitors to be developed as a therapeutic by Santaris Inc. to treat hepatitis C virus (HCV) infection [81–83]. After positive phase 2 outcomes showing safety and reduced HCV viral load, antimiR122 (Miravirsen) is now in long-term phase 2 trials to test the outcome of blocking miR-122 in patients who do not respond to traditional interferon therapy. Moreover, Regulus Therapeutics recently reported that their anti-miR122 inhibitor achieved major reductions in HCV viral load after just a single injection, and has begun phase 2 trials that are expected to be complete in 2016. These positive results suggest that miR-122 stands to become the first miRNA-based therapeutic approved for clinical use. miRNA mimics present a more challenging case for therapeutic targeting, as these are typically double-stranded and susceptible to degradation and detection by the immune system [84]. Therefore, miRNA mimics (or miRNA ‘replacement therapy’) often require a vesicle carrier to enable uptake by the target tissue. Although this approach is more challenging, therapeutic miRNA mimics are nonetheless being developed for treating diseases like cancer, where miR-34a mimics are in phase 1 trials by Mirna Therapeutics using liposomal nanoparticle formulation to suppress solid tumors and hematological malignancies [85]. There have been additional advances in the clinical delivery of anti-sense RNA, with the development of GalNAc-conjugated siRNAs by Alnylum Pharmaceuticals that have high potency and long-term target silencing [86]. These technologies could be applied to assist in the delivery of double-stranded miRNA mimics and accelerate their progression into the clinic. An important consideration for therapeutic miRNA inhibitors and mimics going forward will be their capacity to target a certain cell type or tissue. For atherosclerosis, this will mean targeting macrophages within the atherosclerotic plaque. Systemic delivery of anti-miRNAs generally results in significant portion of the antisense targeting the liver, kidney and spleen, with limited uptake by other tissues [87]. For certain miRNAs with dual roles in targeting cholesterol efflux and HDL biogenesis in both macrophages and the liver, this may have its advantages. However, as we learn more about miRNA function using knockout mice and different methods of inhibition, there will likely be a need to limit the miRNA inhibition to the macrophages only. For example, miR-33 inhibition results in both HDL biogenesis by the liver and increased cholesterol removal from atherosclerotic plaques; however, there could be untoward effects on triglyceride production and hepatic lipid accumulation that could hamper the safety of systemically inhibiting miR-33 [88,89]. Similarly, miR-155 has multiple roles regulating inflammation in different cell types, and inhibiting miR-155 may indeed reduce inflammation with the atherosclerotic plaque. However, given the risk of impaired arteriogenesis following a myocardial infarction, global miR-155 inhibition will not be suitable for clinical development [54]. Nanoparticle delivery of miR-155 inhibitors have shown promise in the aggressive treatment of lymphoma/leukemia in vivo using a much lower dose of anti-miRNAs (~ 50 fold less) [84,90]. It is possible that this cell-specific targeting of miR-155 could be extended to plaque macrophages in the future. Indeed, nanoparticles have been developed that specifically deliver miRNA mimics to atherosclerotic endothelial cells and macrophages in Apoe −/− mice, requiring a 5-fold lower concentration than needed with unconjugated mimics [91,92]. Recently, the delivery of miR-126-3p via ultrasound-targeted microbubble destruction (UTMD) in a chronic nonischemic hindlimb skeletal muscle model promoted angiogenesis and suggests that UTMD could be a novel way to direct miRNA delivery to specific tissues [93]. In the vessel wall, anti-miR21 coating on a bare metal stent in a humanized animal model of vascular injury resulted in the delivery of anti-miR21 to arterial SMCs and the reduction of pathological SMC proliferation and neointima formation [94]. Given that miR-21 is highly expressed in many tissues, including liver, heart, lung and kidney, and is associated with pathological conditions ranging from cardiac remodeling to cancer [95], systemic anti-miR21 therapy may have many

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unwanted side effects and thus local vascular delivery may have greater promise for therapeutic applications [96]. RNA technology is continuing to advance with new methods such as miRNA-dendrimer particles to enhance stability and targeted delivery and is showing very promising pre-clinical results in animal models of aggressive tumors [97]. Overall, cell- and tissue-specific delivery of miRNA therapies in pre-clinical models will help to guide the future understanding of how these therapies can be more targeted and specific, while avoiding any unwanted systemic side-effects, and will undoubtedly accelerate some of these therapies into clinical tools. Anti-sense RNA technology recently hit a milestone with the FDA approval of Mipomersen/KYNAMRO, an anti-sense oligonucleotide against apolipoprotein B, to treat familial hypercholesterolemia (FH) [98,99]. Other anti-sense therapies in advanced clinical development include targeting ApoCIII (phase 2, Ionis Pharmaceuticals) and Lp(a) (phase 2/2a, Ionis Pharmaceuticals; https://clinicaltrials. gov) [100]. Each of these RNA-based therapies is predicted to have significant impact on atherosclerosis development as they target lipids that are highly associated with cardiovascular risk. While these advanced trials will certainly pave the way for future RNA-based therapeutics to reduce cardiovascular disease burden, they are still a long way from targeting the dysregulated pathways specifically within macrophage foam cells that ultimately propagate lesion expansion. 7. Concluding remarks Macrophage cholesterol homeostasis involves multiple intersecting pathways that are under both transcriptional and post-transcriptional control. The interaction between cholesterol accumulation and inflammatory activation means that therapeutic targeting of macrophage foam cells will ultimately reduce inflammation in the vessel wall and thus reduce atherosclerotic lesion burden. The development of miRNA inhibitors and mimics has enabled the rapid growth in our understanding of miRNAs and their targets in health and disease. However, we have only uncovered the tip of the iceberg and there are many intrinsic mechanisms that regulate miRNA function that have yet to be elucidated. A better understanding of the miRNAs that control macrophage cholesterol efflux and inflammation combined with the ability to target these miRNAs locally will offer a major opportunity to combat the complications associated with atherosclerotic vascular disease that are on the rise worldwide. Transparency document The Transparency document associated with this article can be found, in online version. Acknowledgments This work was supported by funding to K.J.R. from the Canadian Institutes of Health Research (MOP130365, MSH130157). References [1] M. Esteller, Non-coding RNAs in human disease, Nat. Rev. Genet. 12 (2011) 861–874. [2] S.L. Ameres, P.D. Zamore, Diversifying microRNA sequence and function, Nat. Rev. Mol. Cell Biol. 14 (2013) 475–488. [3] S. Jonas, E. Izaurralde, Towards a molecular understanding of microRNA-mediated gene silencing, Nat. Rev. Genet. 16 (2015) 421–433. [4] A.E. Pasquinelli, MicroRNAs and their targets: recognition, regulation and an emerging reciprocal relationship, Nat. Rev. Genet. 13 (2012) 271–282. [5] S.M. Mah, C. Buske, R.K. Humphries, F. Kuchenbauer, miRNA*: a passenger stranded in RNA-induced silencing complex? Crit. Rev. Eukaryot. Gene Expr. 20 (2010) 141–148. [6] K.J. Moore, I. Tabas, Macrophages in the pathogenesis of atherosclerosis, Cell 145 (2011) 341–355. [7] D.J. Rader, A.R. Tall, The not-so-simple HDL story: is it time to revise the HDL cholesterol hypothesis? Nat. Med. 18 (2012) 1344–1346.

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Please cite this article as: D. Karunakaran, K.J. Rayner, Macrophage miRNAs in atherosclerosis, Biochim. Biophys. Acta (2016), http://dx.doi.org/ 10.1016/j.bbalip.2016.02.006

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Please cite this article as: D. Karunakaran, K.J. Rayner, Macrophage miRNAs in atherosclerosis, Biochim. Biophys. Acta (2016), http://dx.doi.org/ 10.1016/j.bbalip.2016.02.006