An update on the roles of immune system-derived microRNAs in cardiovascular diseases

2.1 miRNA-17-92 cluster

The cluster encodes six unique miRNAs: miR-17, miR-18a, miR-19a, miR-20a, miR-19b-1, and miR-92a-1 and plays multiple, non-redundant roles in B cell development, supportive of its actions as an oncogene promoting B cell lymphomas.14 Other critical roles include regulation of T cell activation, maturation and development of T-helper subsets (Th) and T_REGS, and additional important functions in monocyte differentiation to macrophages and macrophage activation pathways.15

2.2 miRNA-21

Expressed in multiple cell types in addition to leukocytes, up-regulation of miR-21 (referring to the predominantly expressed mature strand miR-21-5p) is associated with exposure to inflammatory stimuli and immune cell activation.16 Additionally, miR-21 functions as an oncogene and its overexpression results in B cell lymphoma in mice,17 supporting the association of miR-21 with the development of many cancers.18 Aging is associated with increased expression of miR-21 in T cells, resulting in failure to generate memory T cells, contributing to aging-related immune system dysfunction (immunosenescence).19

2.3 miRNA-142

miRNA-142 is a highly evolutionarily conserved miRNA, widely considered to be haematopoietic-lineage specific or enriched.20^,21 Expression of mature isoforms of this miRNA from both arms of the miR-142 precursor (miR-142-3p and miR-142-5p) has been extensively validated. Multiple roles in the development of haematopoietic lineages and immune system function are attributed to these molecules.22–28 The regulation of lineage-enriched expression of miR-142 isoforms may be related to the association of MIR142 promotor activity with super-enhancers which are predominantly active in haematopoietic and immune cell types29 and epigenetic factors, such as lineage and/or context-specific differences in DNA methylation state of the MIR142 promotor.30^,31

2.4 miRNA-146a

miRNA-146a is a powerful negative regulator of both innate and adaptive immune responses.32–34 Expression of miR-146a is driven by the NF-kB signalling pathway35 and is correlated with immune cell activation via T cell receptor signalling or Toll-like receptor activation. Critically, miR-146a-5p functions as a negative feedback regulator, down-regulating NF-kB signalling transducers IRAK1 and TRAF6 to limit cellular activation and its own expression levels.33^,34

2.5 miRNA-155

miRNA-155 is a master regulator of immune system responses. Mice deficient for miR-155 have severely defective lymphoid and myeloid cell development and homoeostasis.36^,37 Activation of immune cells is additionally promoted by miR-155.38 Often, this has been linked to repression of pathways involving the canonical miR-155-5p target, suppressor of cytokine signalling 1 (SOCS1), a potent inhibitor of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signalling pathways.39–41

2.6 miRNA-181

Demonstration that miR-181 is highly and specifically expressed in haematopoietic progenitor cells gave one of the first indications that miRNAs might play critical roles in immune cell lineage differentiation.20 Subsequently, miR-181 family members (miR-181a-1, miR-181a-2, miR-181b-1, miR-181b-2, miR-181c, and miR-181d) have been shown to play critical roles in lymphoid and myeloid lineage differentiation and act as a metabolic rheostat for the immune system.42–44 Down-regulation of miR-181 family member expression may underlie reduced or dysregulated immune system function associated with aging and immunosenescence.45^,46

2.7 miRNA-223

Particularly critical in the regulation of innate immunity,47^,48 expression of miR-223 within the immune system is restricted largely to myeloid-derived cell types, in which miR-223-3p acts as a critical mediator of both myeloid cell differentiation and activation.47

3.1 Hypertension

While immune cell contribution to hypertension was discovered many years ago, it was not until the mid-2000s that a ground-breaking study by Guzik et al. showed that Rag1^−/− mice lacking T and B cells were protected against angiotensin II (Ang-II) and deoxycorticosterone acetate salt-induced hypertension. Moreover, the adoptive transfer of T but not B cells was able to restore the hypertensive abnormalities seen in the control groups, suggesting that T cells are critical mediators of hypertension.49

In a murine model of Ang-II-induced hypertension, levels of the miR-199/214 family member miR-214-3p were up-regulated eight-fold in perivascular tissue of Ang-II-infused mice compared to wild type (WT) mice. Moreover, under Ang-II infusion, miR-214^−/− mice showed blunted hypertension, vascular stiffening, and perivascular fibrosis when compared with WT controls. These effects mapped specifically to changes in the T cell transcriptome in the miR-214 ^−/− mice, which led to blunted T cell activation and infiltration into perivascular adipose tissue (AT). To assess the translational relevance of this, it was found that plasma miR-214-3p expression levels were increased in hypertensive patients.50 In another murine study of Ang-II-induced hypertension, the knockout of miR-31 (miR-31^−/−), which maps specifically to T_reg and Th₁₇ cells led to blunted hypertension and decreased cardiac hypertrophy and fibrosis. Furthermore, miR-31 ablation in mice resulted in a decrease of kidney infiltrating CD45⁺F4/80⁺ macrophages and an increase in kidney specific CD4⁺FoxP3⁺T_REGS. As a result, miR-31^−/− mice had reduced vascular and renal damage. These differences mapped specifically to miR-31^−/− mediated post-transcriptional regulation of its direct target protein phosphatase 6c.51

3.2 Obesity and epicardial AT

Obesity and diabetes are major risk factors for CVD. AT is an endocrine organ of paramount importance in the development of systemic inflammation, which affects cardiovascular cell metabolism, function, and disease progression. Furthermore, the immune system plays critical roles in the development and function of AT and obesity is increasingly considered to be an immunometabolic disease. Thickening of epicardial adipose tissue (EAT), which surrounds the heart and major vessels is associated with coronary artery disease (CAD). In patients with sudden-CAD associated death, levels of miR-34a-3p and miR-34a-5p in EAT were up-regulated.52 This is of particular interest as miR-34a has been associated with obesity-induced systemic inflammation via the suppression of anti-inflammatory ‘M2’ macrophage polarization and increase in pro-inflammatory ‘M1’ macrophages and cytokines within visceral AT of murine models of diet-induced obesity.53 Furthermore, whole-genome sequencing on EAT from CAD patients, shows that genes involved in antigen presentation, chemokine signalling, and systemic inflammation are up-regulated, whilst levels of miR-103-3p, which regulates Th₂ chemokines, like CCL13, were down-regulated.54 Interestingly, the presence of EAT around the left atria (LAEAT) has been linked to the prevalence of atrial fibrillation. Using data from the miRhythm study and bioinformatic platforms, Tran et al. analysed miRNA levels associated with LAEAT from AF patients. Levels of miR-155-5p and miR-302a-3p were associated with LAEAT in AF. Both miRNAs are involved in the regulation of IL-8, which has been linked to leukocyte trafficking and activation.55^,56 Furthermore, downstream predicted genes of both miRNAs were associated with cardiac hypertrophy and adipogenesis.4

4.1 Atherosclerosis

Atherosclerosis is considered to be an inflammatory, immune-mediated disease. Of the immune cells associated with atherosclerosis pathogenesis, macrophages play a central role in plaque formation and disease progression. As LDL retained beneath the endothelial wall becomes oxidized (oxLDL), it activates ECs and promotes the recruitment and infiltration of monocytes/macrophages. Uptake of oxLDL and internalization of apoptotic cell fragments by macrophages results in lipid accumulation and foam cell (FC) formation. The roles of miRNAs in the regulation of macrophages during atherosclerosis have been extensively investigated.57 Recent work has continued to uncover novel miRNA-mediated mechanisms of macrophage regulation and their athero-protective and atherogenic effects (Figure 2). Utilizing publicly available miRNA array data, Zheng et al.58 postulated that miR-133b-3p expression in macrophages serves a pro-atherogenic role by modulating expression of mastermind-like protein 1 (MAML1), tentatively supporting studies which highlight a role for NOTCH signalling in plaque leukocyte influx and atherosclerosis progression.59–61 However, although the authors convincingly demonstrated regulation of MAML1 by miR-133b-3p, given the well-appreciated role of MAML1 as a transcriptional co-activator of NOTCH signalling, it is not immediately clear how de-repression of MAML1 expression via down-regulation of miR-133b-3p might promote the associated down-regulation of various NOTCH pathway components—as implied by the authors. Therefore, further work which clarifies the exact relevance of the miR-133b–3p/MAML1 interaction in macrophages during atherosclerosis is required.

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miRNA-17-5p—a potentially useful, circulating biomarker for monitoring atherosclerosis severity,62 has also been implicated in the regulation of inflammation and macrophage lipid accumulation during atherosclerosis, through the direct regulation of ATP-binding cassette transporter A1 (ABCA1), a critical component of cellular reverse cholesterol transport/lipid efflux.63 Down-regulation of ABCA1 leads to accumulation of intracellular lipids, promoting FC generation during atherosclerosis. miRNA-17-5p joins several additional miRNAs demonstrated to regulate ABCA1 in macrophages, thereby limiting reverse cholesterol transport,57^,64 including the miR-33 family.65^,66 miRNA-33-5p also regulates macrophage expression of peroxisome proliferator-activated receptor gamma co-activator 1-alpha, a master regulator of mitochondrial biogenesis and energy metabolism—therefore limiting cholesterol efflux via ABCA1 by restricting production of ATP.67 Inhibition of cholesterol efflux can promote formation of lipid-rich cell membrane rafts, promoting pro-inflammatory extracellular signalling pathways and inflammation.68 Alongside enhanced plaque instability, miR-205-5p was recently shown to direct lipid raft formation in macrophages, promoting pro-inflammatory macrophage phenotypes,69 expanding the understanding of miRNA-mediated control of macrophage lipid transport and its importance during atherosclerosis.

Defective macrophage clearance of other apoptotic macrophages and FCs (efferocytosis) may also underlie inflammation and atherosclerosis progression. Down-regulation of miR-378a-3p in aortic macrophages of hyperlipidemic Apoe^−/− mice was shown to result in de-repression of signal regulatory protein α (SIRPα),70 the transmembrane signalling receptor for CD47 (integrin-associated protein). The CD47: SIRPα axis act as a don’t eat me signal and up-regulation of this pathway prevents clearance of apoptotic cells within plaque lesions, accelerating atherosclerosis progression. Dysregulation of the CD47: SIRPα axis could also underlie atherosclerosis progression in humans, as was implied by the observation that the long non-coding RNA (lncRNA) myocardial infarction associated transcript (MIAT) was significantly higher in the circulating sera of atherosclerosis patients and those with symptoms of vulnerable atherosclerotic plaque, compared to controls, indicating that MIAT may also serve as a useful biomarker of plaque stability and atherosclerosis severity.71 In this study, the authors suggest that MIAT acts as a competing endogenous RNA (ceRNA) for miR-149-5p in macrophages and contains complementary miR-149-5p binding sequences which allow MIAT to ‘sponge’ miR-149-5p away from CD47, de-repressing CD47 expression. This study appears to convincingly demonstrate a functional role for this interaction, using ceRNA overexpression systems and in vitro miRNA manipulation to probe the nature of this dynamic in macrophage cell lines. However, the degree to which ceRNAs truly act to regulate endogenous miRNA activity through modulation of bioavailability for molecular target interaction is controversial (reviewed by Thomson and Dinger72). In their article, the authors do not investigate the limitations that physiological expression levels of both molecules may impose upon the outcome of the interaction during the real-world context of disease. This general criticism applies to many attempts to characterize cell-endogenous physiological regulators of miRNA activity. However, the concept presented in this work highlights the importance of considering the complex components of networks that regulate the functional activity of specific miRNAs in a given cell type.

Additional leukocytes also serve critical roles in the pathophysiology of atherosclerosis. T cells and particularly the CD4⁺ lineage are important players in atherosclerosis pathogenesis and the influx of CD4⁺ T cells to the vascular walls may be regulated by alterations to cytoskeleton dynamics as a result of aberrant expression of miR-142-3p.73 CD4⁺ Th₁ responses, directed by the Th₁ master T-box transcriptional regulators T-BET and (to some extent) Eomesodermin (EOMES) are the most prevalent Th subset in atherosclerotic plaques and promote atherosclerosis progression and severity.74–76 miRNA-29 seed-family members miR-29a-3p and miR-29b-3p act to restrict T-BET and EOMES expression in T cells77 and may therefore putatively alleviate atherosclerosis and enhance plaque stability by directly inhibiting Th₁ development. However, evidence that global antagonism of miR-29 members using locked nucleic acids (LNAs) decreases atherosclerotic lesion size and promotes plaque remodelling and stability in mice,78 suggests that in certain cell types, miR-29 family members may actually contribute to disease progression. However, in this study, the impact of LNA-miR-29 on the development of Th1 responses was not specifically addressed. More studies are required to fully define the contribution of T-cell-derived miR-29 family members to the promotion of plaque-associated Th₁ development, both during human and murine disease, in order to guide potential therapeutic strategies.

4.2 Myocardial infarction

Monocytes and macrophages are critical mediators of inflammation and promote cardiomyocyte apoptosis during the early inflammatory response post-MI, but also contribute to later stages of left ventricular remodelling and dilation, eventually leading to HF.79 miRNA-21-5p may be a critical endogenous mediator, which limits the polarization of pro-inflammatory macrophages in the heart, post-MI. miRNA-21-5p expression was up-regulated rapidly in infarct areas of murine cardiac tissue, however, MI-induction in mice deficient for miR-21 (miR-21^−/−) resulted in worse survival rates and greater infarct size and scarring.80 Macrophage expression of miR-21-5p directly restricted inflammatory activation and cytokine production by targeting Kelch repeat and BTB (POZ) domain containing 7 (KBTBD7), a transcriptional activator involved in mitogen-activated protein kinase signalling. In the absence of miR-21-5p, KBTBD7 overexpression enhanced macrophage activation by promoting p38 and NF-kB activation.80 Promotion of miR-21 regulatory mechanisms through intravenous delivery of nanoparticles (NPs) containing miR-21-5p mimics, targeted to cardiac macrophages shortly after MI-induction may therefore be a therapeutic strategy to limit pro-inflammatory/promote reparative, wound healing macrophage phenotypes, leading to attenuation of pathological remodelling.81 Therapeutically targeting miR-375-3p in macrophages may be an additional means of modulating pro-inflammatory macrophage development post-MI, via activation of 3-phosphoinositide-dependent protein kinase 1 and reduction of pro-inflammatory cytokine expression.82 Additionally, expression of a superoxide producing enzyme, NADPH oxidase 2 (NOX2) is enhanced in cardiomyocytes and macrophages post-MI83 and promotes macrophage inflammatory polarization and cytokine expression84 and is associated with adverse cardiac remodelling post-MI.85 In both human and murine macrophages, miR-106b-5p, miR-148b-3p, and miR-204-5p were shown to suppress NOX2 and targeted up-regulation of these miRNAs through pH-sensitive polyketal NP-specific delivery to macrophages significantly improved infarct size and cardiac function post-MI in mice, highlighting another potential therapeutic axis for management of MI86 (Figure 3A).

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4.3 Myocarditis

Myocarditis is an important cause of sudden cardiac death and heart failure, particularly in young, otherwise healthy patients. A breakdown in tolerogenic immune mechanisms is apparent during myocarditis and may particularly underpin autoimmune-mediated myocarditis. For example, miR-98-5p expression is enhanced in B cells during experimental autoimmune myocarditis (EAM) of mice and acts to suppress regulatory IL-10 expression.87 Likewise, polarization of tolerogenic dendritic cells, which promote induction of T_REGS, may be inhibited in EAM as a result of miR-223-3p down-regulation, de-repressing NLRP3 inflammasome expression.88 However, the study of DCs in this work relies entirely on the use of murine bone marrow-derived dendritic cells generated through in vitro culture, potentially limiting conclusions regarding translational relevance in humans. None-the-less, T_REGS are generally thought to be cardioprotective during myocarditis while effector CD4⁺ T cells are critical players in myocarditis pathogenesis.89 Th₁₇ effectors play a particularly pathogenic role in later or chronic stages of autoimmune-related myocarditis progression and in the progression to dilated cardiomyopathy (DCM), while the balance of Th₁₇ to T_REG cells also serves to modulate myocarditis severity, with reduced T_REGS leading to more profound Th₁₇ responses and more severe disease.89 As reported for inflammatory conditions of other solid organs,39 miR-155-5p plays a critical role in regulating Th₁₇/T_REG balance during murine EAM, in which up-regulation of miR-155-5p in cardiac CD4⁺ T cells following induction of EAM promotes Th₁₇ responses, while inhibiting generation and responses of T_REGS.90 Additional evidence from Coxsackievirus-B3 murine models of viral myocarditis also supports a pathogenic role for up-regulated miR-155-5p in dysfunctional activation of T cells during acute myocarditis, as well as in promoting monocyte/macrophage cardiac infiltration and inflammatory ‘M1’ cardiac macrophage polarization.91^,92 Moreover, up-regulated expression of miR-155-5p, as well as the immune-related miRNAs miR-146b-5p and miR-21-5p were observed in human ventricular septal biopsies of acute viral myocarditis patients.91 Targeting miR-155-5p expression may therefore hold therapeutic promise for the treatment of myocarditis in certain settings. However, a pathogenic role for miR-155-5p during myocarditis may not be absolute. Natural Killer (NK) cells are innate mediators of viral clearance and are protective in viral myocarditis, with roles in restricting autoimmune inflammation during EAM also reported.93 NK cell activation is positively correlated with expression of miR-155-5p, and promotes NK cell expression of IFNγ, partially through repression of the inositol phosphatase SHIP1.94 During murine viral myocarditis, increased NK expression of miR-155-5p and enhanced NK cell activation is apparent—promoting viral clearance and reducing cardiac immune cell infiltration and inflammatory cytokine production95 (Figure 3B).

4.4 Cardiac hypertrophy

Leukocyte infiltration into cardiac tissue exacerbates cardiovascular dysfunction in in vivo models of pressure overload-induced hypertrophy. miRNA-155-5p was increased in cardiac biopsies of hypertrophic aortic stenosis patients and correlated negatively with cardiac function yet positively with wall thickness relative to non-hypertrophic controls undergoing coronary bypass artery grafting.41 Furthermore, in two different murine models of pressure overload, genetic ablation, or pharmacological inhibition of miR-155-5p led to a decrease in cardiac infiltrating leukocytes, particularly CD68⁺CD11b⁺F4/80⁺ monocytes/macrophages. This was accompanied by preserved cardiac function and the absence of hypertrophy, both at the physiological and gene level. These affects were shown to be macrophage specific, as adenoviral-mediated inhibition of miR-155-5p in cardiomyocytes of WT mice did not prevent hypertrophy, whilst in bone marrow adoptive transfer experiments, the presence of miR-155^−/− macrophages in WT mice was able to protect against hypertrophy. SOCS1 was the main target of miR-155-5p in macrophages postulated to drive hypertrophy; in the absence of miR-155-5p, SOCS1 overexpression inhibited the JAK/STAT signalling axis and specifically inhibited STAT3 signalling, suppressing the macrophage inflammatory response and release of paracrine mediators driving myocyte hypertrophy.41 See Figure 3C.

4.5 Heart failure

An ongoing inflammatory response is associated with chronic HF. HF patients have elevated levels of circulating pro-inflammatory cytokines including IL-6, IL-1β, and TNFα, and recent analysis of the results of the CANTOS phase III clinical trial revealed that use of the anti-IL-1β monoclonal antibody canakinumab (Ilaris), reduced hospitalizations and mortality related to HF in MI-patients.96 However, other phase III trials aimed at modulating HF with anti-inflammatory interventions have had less successful outcomes, suggesting that inflammatory responses may only drive HF in certain patient cohorts.97 In DCM patients, CD4⁺ T cell over-activation and proliferation is associated with down-regulation of miR-451a-5p and increased expression of its target Myc,98 marking the importance of T cell responses and their regulation by miRNAs in the development of HF. Immunosenescence is also related to the development of HF associated with aging. Ageing-associated miRNAs, such as miR-181 family members—which are important positive regulators of B-cell lymphopoiesis,20 are reduced in immune cells of aged patients45 and miR-34a-5p which inhibits B cell maturation and demonstrates enhanced expression in leucocytes with aging99^,100 may underlie these effects.46 Targeting these molecules may mitigate aging-associated HF progression by restoring lymphocyte function. See Figure 3D.

4.6 Vasculitis

Vasculitis covers a highly heterogeneous group of multi-system autoimmune, inflammatory conditions that affect blood vessels. Although rare, complications of vasculitides can include systemic emboli, thrombosis, aneurysms, sepsis, vessel occlusion, and major end-organ damage. In Wegener’s granulomatosis—the most common form of anti-neutrophil cytoplasmic autoantibody-associated vasculitis, accumulation of effector CD4⁺ T cells and a T-helper profile skewed towards IL-17 and IL-23 producing Th₁₇ cells101–103 alongside a reduction in T_REGS suppressive capacity104^,105 is thought to critically regulate disease pathogenesis. In this setting, aberrantly up-regulated expression of miR-142-3p may underlie defective T_REGS suppressive activity, potentially driving pathogenic effector T cell responses. miRNA expression profiling of a circulating memory-like population of T_REGS found to be enriched within the CD4⁺ T cell pool of patients relative to healthy controls revealed miR-142-3p to be significantly up-regulated alongside decreased expression of the miR-142-3p target adenylate cyclase 9 (ADCY9) and a corresponding reduction in intracellular cyclic adenosine monophosphate (cAMP)106—a central mediator of T_REG suppression of effector T cells, synthesized by ADCY9. Dysregulated T_REGS activity alongside aberrant changes in T_REGS miRNA expression has also been reported in Kawasaki’s disease (KD)—an acute form of systemic vasculitis, which can affect the coronary arteries. The proportion of circulating T_REGS and T_REGS expression of markers of maturation and suppressive function were significantly lower in KD patients (aged 3–54 months), while miR-155-5p and miR-21-5p expression was reduced and miR-31-5p expression was increased significantly.107 Putatively, these miRNA expression changes during KD lead to reduced proportion and activity of T_REGS through de-repression of the miR-155-5p target SOCS1-inhibiting STAT5 signalling critical for T_REGS homoeostasis and function, and restricted expression of the T_REGS master transcription factor FOXP3.107^,108