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Small but smart—microRNAs in the centre of inflammatory processes during cardiovascular diseases, the metabolic syndrome, and ageing

Blanche Schroen, Stephane Heymans
DOI: http://dx.doi.org/10.1093/cvr/cvr268 605-613 First published online: 12 October 2011

Abstract

With a progressively growing elderly population, ageing-associated pathologies such as cardiovascular diseases are becoming an increasing economic, social, and personal burden for Western societies. Interestingly, all ageing-associated diseases share a common denominator: inflammation. Recently, microRNAs were shown to be implicated in the full range of processes of ageing, inflammation, and cardiovascular diseases. This review focuses on their role in cardiovascular diseases with emphasis on their implication in the inflammatory processes that accompany heart failure, atherosclerosis, coronary artery disease, and finally obesity and diabetes as components of the ageing-associated metabolic syndrome.

  • MicroRNA
  • Inflammation
  • Cardiovascular disease
  • Metabolic syndrome
  • Ageing

1. Introduction

The incidence of cancer and cardiovascular diseases in Western societies is rising due to the ageing of the population. While cancer is seen as a ‘gain-of-function’ disease, in which cells adapt novel mechanisms to survive, cardiovascular diseases are of the ‘degenerative’ type, in which cells become increasingly dysfunctional and as a consequence tissue functions decline.1 Interestingly, all ageing-associated diseases share a common denominator: inflammation (Figure 1).2 Here, we must distinguish acute from chronic inflammation, where the chronic type is associated with the ageing process. While acute inflammation involves immune cell influx to address injury or infection, chronic inflammation does not only involve the presence of immune cells but more importantly it is a state in which cells such as fibroblasts, epithelial, and endothelial cells (ECs) enter a senescent state and produce inflammatory mediators, changing the tissue microenvironment.2,3 Cellular senescence is at the basis of the chronic inflammatory state during ageing, and is characterized by the mitotic exit of dividing cells while they remain metabolically active. Cellular senescence is thought to be a major player in the cardiovascular ageing process.4

Figure 1

Guide to the build-up of the review. The processes of ageing and chronic inflammation are indistinguishably intertwined. Both entities are also crucially associated with HF, atherosclerosis, and the metabolic syndrome, which all have a major inflammatory component and which incidence rises dramatically in the elderly. Inflammation-related microRNAs have a clear role in these ageing- and inflammation-associated pathologies (chapters 1–3; shown in grey) and also appear in the circulation (chapter 4; shown in red). In addition, ageing-associated as well as immune-associated microRNAs are often regulated in HF (chapter 5; Tables 1 and 2; microRNAs highlighted in bold).

MicroRNAs have been implicated in the full range of processes of cellular senescence, inflammation, and cardiovascular diseases.57 They are the most studied class of non-coding RNA molecules so far. This review focuses on their role in cardiovascular diseases with emphasis on their implication in the chronic inflammatory processes that accompany heart failure (HF), atherosclerosis, and coronary artery disease (CAD). Inflammation and cardiovascular morbidity converge into the process of ageing, or vice versa, ageing accelerates inflammation, and cardiovascular diseases. The metabolic syndrome with diabetes, obesity, and hyperlipidaemia as a trigger for inflammation, HF, and atherosclerosis will be addressed. The role of microRNAs in the inflammatory components of these pathologies will be subject of the next paragraphs. The organization of the review is outlined in Figure 1.

2. Role of inflammation-related microRNAs in HF

Recent advances in microRNA research technologies have ensured major progress in our understanding of the role of myocytic and fibrotic microRNAs in the development of HF (reviewed elsewhere in this issue). In contrast, only one study describes a role for a microRNA in inflammation during myocardial infarction (MI)-induced HF. Using computational analyses elegantly integrating microRNA with mRNA expression data, Zhu et al.8 found a role for miR-98 in MI, during which it is a central hub in inflammation. In brief, they generated cardiac microRNA and mRNA expression data of MI in rats, built regulatory networks taking into account gene abundance, and searched these networks for functional enrichment. In this way, miR-98 was found to be a potential regulator of inflammatory pathways following MI. However, further in vivo proof is needed to determine whether miR-98 function indeed is central in the inflammatory response during MI. Interestingly, but not surprisingly, these authors found gene abundance to have an impact on the regulatory performance of a microRNA using this in silico approach, a modality that already has been recognized in the early days of prediction of microRNA–mRNA interactions.9 For the integration of microRNA with mRNA expression data, this is a factor to take into account.

To conclude, a role for microRNAs in the inflammatory response during HF is still open to discoveries. On the other hand, there are a few studies implicating inflammatory microRNAs in the development of specific forms of HF (Figure 1), focusing on their role in modulating myocyte function.

MiR-146a is a well-known player in the immune system. Its expression in inflammatory cells is induced by lipopolysaccharide and is nuclear factor-kappaB (NF-κB)-dependent, and it acts as a negative feedback regulator of the innate immune response by targeting the proinflammatory adapter proteins TNF receptor-associated factor 6 and interleukin-1 receptor-associated kinase 1.10 MiR-146a expression is increased in doxorubicin-induced HF. These authors show that it targets the v-erb-a erythroblastic leukaemia viral oncogene homolog 4 (ErbB4) in the cardiac myocyte and may thereby cause cardiac myocyte dysfunction.11 However, in vivo knockout or antimiR studies in doxorubicin-induced HF or other forms of HF still lack.

Another example is miR-223, which was originally described as a myeloid lineage-specific microRNA.12 MiR-223 may modulate diabetic HF by regulating glucose metabolism in cardiac myocytes.13 The miR-223 target MADS box transcription enhancer factor 2c (Mef2c) is not involved in this cardiac process, while the glucose transporter Glut4 was found to be induced by cardiac myocyte miR-223, facilitating myocytic glucose uptake. MiR-223 was also found up-regulated in atrial biopsies of patients with atrial fibrillation and rheumatic heart disease, a complication of the autoimmune disease rheumatic fever.14

Another well-known central regulator of immune activation is miR-155.15 A polymorphism in its target gene angiotensin II type 1 receptor (AT1R) is associated with increased risk of adverse outcome in hypertensive subjects.16,17 However, the implication of miR-155 in HF, possibly via its target gene AT1R, which has major roles in cardiovascular as well as immune cell functions, is unknown. Finally, miR-21 has a role in multiple pathologies including cardiovascular diseases and inflammation,18 and was recently extensively studied for its function in cardiac fibrosis and concomitant HF.19 The field is awaiting first data on real genetic evidence for the role of immune cell-derived microRNAs in mediating the cross-talk between inflammatory cells and the different cardiac cells during HF. No publications this date exist on the role of microRNAs in acute viral myocarditis, or in chronic inflammation affecting diabetic, ischaemic, hypertensive, or autoimmune heart disease. An unmet medical need exists to develop novel RNA-based therapies specifically targeting the uncontrolled inflammatory reaction in viral or autoimmune myocarditis, the most aggressive form of inflammatory heart disease affecting young previously healthy individuals.20 MicroRNA-based medicines may also help dampen the chronic inflammatory processes in the more ageing-dependent cardiovascular disease processes caused by diabetes, long-term smoking, hyperlipidaemia, and hypertension.

3. MicroRNAs as regulators of the inflammatory response during atherogenesis

3.1 MicroRNAs modulate inflammatory function of ECs

Inflammation is a hallmark of all stages of atherosclerosis, whereby the initial stage is characterized by leucocyte recruitment to activated ECs.21 Interestingly, microRNA-deficient ECs, following Dicer knockdown, showed proliferative defects22,23 and were hallmarked by decreased levels of inflammatory chemokines and cytokines, including interleukin (IL)-8, IL-1β, and chemokine (C-X-C motif) ligands (CXCL) 1 and 3, suggesting that microRNAs play a central role in EC function.23 Restoration of levels of microRNAs -221 and -222, which are among the highest expressed microRNAs in ECs,24 could not restore the proliferative capacity of Dicer-deficient ECs.23

Monocyte adhesion upon EC activation is a critical step in inflammatory invasion of an atherosclerotic lesion.21 Several microRNAs are differentially expressed in activated ECs (Figure 1) and modulate adhesion molecule expression, pinpointing their central role in atherogenesis; i.e. in human umbilical vain ECs (HUVECs), miR-126 was found highest expressed by microarray analysis, and inhibited leucocyte adherence through the direct regulation of the vascular cell adhesion molecule-1 (VCAM-1).24 Also in HUVECs, miR-21 expression increases upon Oscillatory shear stress (used to induce monocyte adhesion to ECs) and this response depends on c-jun/activator protein-1 (AP-1).25 MiR-21 promotes monocyte adhesion by enhancing adhesion molecule expression, including VCAM-1 and monocyte chemotactic protein-1 (MCP-1). MiR-21 also targets peroxisome proliferator-activated receptor alpha (PPARα), and becomes part of a positive feedback loop with lowered PPARα allowing increased AP-1 activity.

MicroRNA array analysis of athero-susceptible vs. athero-protected parts of swine aorta yielded 27 up-regulated miRs (among which are miRs -221 and -21) and 7 down-regulated miRs.26 Here, EC miR-10a is most down-regulated and targets Mitogen-activated protein kinase kinase kinase 7 and β-transducin repeat-containing gene, two factors involved in IκBα degradation. Therefore, decreased miR-10a in athero-susceptible regions allows NF-κB activation and the consequent increased expression of the inflammatory biomarkers MCP-1, IL-6 and -8, VCAM-1, and E-selectin in ECs.

MicroRNA array analysis of HUVECs undergoing OS vs. laminar shear stress resulted in 10 confirmed differentially expressed microRNAs, including miR-1275, -638 and -663 (up) and miR-320a, -b, -c,- 151-3p, -195, -139-5p, and -27b (down).27 The OS-induced regulation of an endothelial microRNA, miR-663, was linked to the induction of pro-inflammatory gene expression and to increased adhesion of monocytes. There was no overlap between this in vitro microarray experiment and the microRNAs regulated in the in vivo model of athero-susceptibility;26 more peculiarly, miRs -27b and -151-3p showed inverse regulations in vivo and in vitro, speculatively due to the mixed cell types in vivo or the artificial nature of culturing conditions in vitro. This reinforces the requirement of in vivo validation experiments to confirm a role for genes found by expression studies.

The inflammation-linked miRs -155 and -221/-222 were found highly expressed in HUVECs and all target v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1), an important endothelial transcription factor that robustly regulates endothelial inflammation, angiogenesis, and vascular remodelling.28 Angiotensin II, used to activate pro-inflammatory signalling by ECs, increased Ets-1 expression, leading to up-regulation of Ets-1 downstream genes, including VCAM-1, MCP-1, and fms-related tyrosine kinase 1 (FLT-1), the vascular endothelial growth factor/vascular permeability factor receptor. MiRs -155 and -221/-222 inhibited monocyte adhesion by targeting Ets-1, while miR-155 inhibited HUVEC migration, possibly by targeting the AT1R. While miR-221 was found up-regulated in athero-susceptible tissue26 and both miR-221 and -222 were significantly higher in endothelial progenitor cells of patients with CAD,29 it is unclear what effect AngII had on miR-155 and -221/-222 expression in HUVECs. However, stimulation of HUVECs with other pro-inflammatory moderators, basic fibroblast growth factor (bFGF) and IL-3, reduced miRs -221 and -222.30 These authors show that miR-222 but not miR-221 inhibited proliferation and migration of stimulated HUVECs, possibly by targeting of signal transducer and activator of transcription 5A, the down-stream signaller used by bFGF and IL-3 to trigger vascular EC morphogenesis. Furthermore, bFGF and IL-3 were both found to decrease miR-126 and -296, and miR-21 and -17-5p showed a trend to increase. All these studies have been done in vitro, excluding their interaction and cross-talk with other cells. The relevance of these findings in vivo in disease models of ischaemia, diabetes, or hypertension has not been addressed yet. In view of previous paradoxical findings in in vitro set-ups compared with in vivo animal studies, extrapolating in vitro findings to the disease process itself has to be done with extreme care.

3.2 MicroRNAs modulate the macrophage response to oxidized LDL

Activation of macrophages with oxidized low density lipoproteins (oxLDL) is used as an in vitro model for macrophages present in atherosclerotic plaques. A first study addressing the role of microRNAs in this system performed microarray analysis on oxLDL-activated human primary peripheral blood monocytes.31 Both miR-125a-3p and -5p were significantly up-regulated. MiR-125a-5p inhibition increased inflammatory cytokine secretion and lipid uptake, possibly via its target Oxysterol binding protein-related Protein 9 (ORP9), which is involved in lipid metabolism and membrane transport.

The inflammatory miRs -146a and -146b were also found up-regulated both by microarray and quantitative PCR in this study.31 Another study, however, showed that miR-146a expression decreased upon oxLDL stimulation of THP-1 macrophages.32 Here, decreased miR-146a in oxLDL-activated macrophages was linked to an increase of its target Toll-like receptor 4 (TLR4), involved in lipid uptake and inflammatory cytokine secretion, thereby allowing the accumulation of oxLDL on the one hand and an inflammatory response characterized by IL-6 and -8, chemokine (C-C motif) ligand 2, and matrix metalloproteinase-9 on the other hand. Thus, miR-146a would be anti-inflammatory in THP-1 macrophages. However, it is striking that the expression pattern of activated THP-1 macrophages is completely the opposite of often-observed changes in miR expression; i.e. miR-21 and -155 are often found up-regulated and miRs -320a-c are found down-regulated in activated cells in an atherosclerotic setting, that is, in activated ECs,2527,30 and in oxLDL macrophages.31,33 The divergence of these data may reflect the dissimilarity of cell types used. Also peripheral blood mononuclear cells (PBMCs) of patients with acute coronary syndrome34 and peripheral monocytes of patients with CAD35 had enhanced miR-146a expression. In CAD, high levels of miR-146a and TLR4 in circulating mononuclear cells were independent predictors of cardiac events after a 12-month follow-up.35 In acute coronary syndrome, miR-146a had a dual function in peripheral monocytes: (i) it increases the transcription factor T-bet thereby leading to increased Th1 differentiation and (ii) it induces pro-inflammatory cytokine (TNFα, MCP-1) and NF-κB p65 production.34 In conclusion, even though the general consensus is that miR-146a up-regulation counterbalances activated innate immunity,3641 it's role in diverse pathological conditions is not always consistent.

Monocytes and macrophages activated with oxidized LDL increased their miR-155 expression.31,33 Inhibiting miR-155 in activated macrophages leads to enhanced uptake of oxLDL and increased expression of scavenger receptors, such as CD36 and CD68, and promoted IL-6, -8 and TNFα cytokine release, presumably via myeloid differentiation primary response gene (88) (MyD88) and NF-κB signalling.33 Therefore, in these atherosclerotic conditions, miR-155 appears to be anti-inflammatory, opposite to the generally accepted association of miR-155 with a pro-inflammatory state.42 However, its implication in vivo in atherosclerosis needs to be addressed, since the function of microRNAs may differ depending on the phenotype of the macrophage, and the influence of these inflammatory cell-mediated microRNAs on neighbouring endothelial or smooth muscle cells.

4. MicroRNAs in the metabolic syndrome

Chronic inflammation is central in the metabolic syndrome, including obesity, type II diabetes mellitus (DM), hypertension, and atherosclerosis.43 The role of microRNAs in the metabolic syndrome and associated aetiologies was reviewed before.44 Here, we go into the link of obesity and diabetes with inflammation and associated microRNAs (Figure 1).

During obesity, the adipose tissue environment is characterized by a chronic inflammatory state, with increased production of the inflammatory cytokine TNFα by macrophages as the factor largely responsible for insulin resistance in obese adipose tissue.43 Interestingly, obesity causes the loss of microRNAs that characterize fully differentiated and metabolically active adipocytes, including miRs -103 and -143. On the other hand, expression of miRs -221 and -222 decreased during adipogenesis and increased in obese adipocytes. This inverse regulation of microRNAs during adipogenesis vs. obesity is likely mediated by high levels of inflammatory TNFα in obese fat tissue, since miRs -103 and -143 were decreased and miRs -221 and -222 were increased by TNFα.43

Obese subjects were found to have a unique microRNA expression profile in omental fat as well as blood, when compared with non-obese individuals, with miRs -17-5p and -132 up-regulated only in fat and in the circulation of obese subjects.45 These two microRNAs also correlated significantly with body mass index (BMI) and fasting blood glucose levels. A role for miR-132 in inflammatory processes during obesity was suggested by an in vitro study using primary human preadipocytes and in vitro differentiated adipocytes, where in response to nutritional availability, induction of miR-132 decreases sirtuin 1-mediated deacetylation of p65 leading to activation of NF-κB and transcription of IL-8 and MCP-1.46

Inflammatory microRNAs also have functions in diabetic conditions. MiR-21 and -146 expression were induced by the proinflammatory cytokines IL-1β and TNFα in pancreatic islets, and inhibition of these microRNAs prevented the reduction in glucose-induced insulin secretion as a result of cytokine exposure.47 In vivo, in diabetic kidney disease, miR-21 levels were increased in renal cortices of type I diabetic mice, leading to decreased levels of phosphatase and tensin homolog (which at normal levels inhibits renal cell hypertrophy and matrix expansion) and increased activity of TOR complex 1, necessary for cellular hypertrophy.48 MiR-221 was induced by high glucose levels in HUVECs, inhibiting c-kit and consequently impairing EC migration.49 Finally, exposure of adipocytes to high glucose levels induced miR-222, among others.50

5. Circulating microRNA profiles of cardiovascular diseases

5.1 Circulating microRNA profiles of cardiac disease

Although there is much to discover about the exact function of circulating microRNAs, their presence in the circulation and association with diverse pathologies is now well-accepted.51 MicroRNAs circulate either in cell-shed microparticles—including apoptotic bodies and exosomes—52, in high density lipoprotein (HDL) particles,53 or as cell-free miRNAs in serum, recently demonstrated to be carried by Argonaute 2.54 The association of circulating microRNAs with cardiovascular disease is reviewed elsewhere in this issue of Cardiovascular Research. Here, we will briefly touch upon the role of circulating microRNAs in cardiovascular diseases with a prominent inflammatory character (Figure 1). The most studied in this respect is MI, which seems to induce a very consistent serum presence of the cardiac-specific microRNAs miR-1, -208, and -499.51,5557 Their unique presence following cardiac damage correlates with clinical markers of damage, including troponin T56 and creatine kinase-MB activity,55 and may even be earlier than troponins.57 This suggests that serum presence of these microRNAs represents passive leakage from damaged cardiac myocytes.

Another typical form of HF with strong immune involvement is viral myocarditis, and we hypothesized the presence of inflammatory microRNAs in the circulation of these patients to increase.56 However, serum presence of leucocyte-associated miRs -146a, -146b, -155, and -223 was not changed in patients with viral myocarditis when compared with controls, despite significant leucocytosis in these patients. On the other hand, cardiac miRs -208 and -499 did show a significant increase in acute viral myocarditis, correlating with the degree of myocyte leakage or death indexed by troponin T release.

The cellular origin of miR-423-5p, which was increased in sera of HF patients, is yet unknown.58 Interestingly, one of the highest detected and up-regulated serum microRNAs in this study, besides miR-423-5p, was the inflammation- and EC-associated miR-221.58

5.2 Circulating microRNA profiles of vascular disease

On the vascular site, CAD and atherosclerosis have a major inflammatory component. An interesting pattern observed in CAD was that highly present circulating microRNAs would decrease in the blood of CAD patients and most of these were EC-derived, including miR-126 and the miR-17-92 cluster (Figure 1).59 Down-regulation of miR-126 in CAD was confirmed by Zampetaki et al.60 Among the down-regulated microRNAs in CAD blood were also miRs -21, -146a, -221, and -223.59 The overlap with a CAD whole blood profiling study with similar patient numbers61 is small; of the 46 listed down-regulated miRs in Fichtlscherer et al.,59 2 were also found down-regulated in whole blood (miRs -20a and -93) and miR-23a even showed an opposite regulation in the two studies. A third study on CAD and microRNAs used PBMCs as source of RNA, and found relatively unknown microRNAs to be involved: miRs -135a and -147.62 Note that in the latter study, only 157 microRNAs were studied. Interestingly, miRs -21 and -223 were seen as housekeeping miRs.

A larger study addressed the presence of a selected set of 13 circulating microRNAs in 104 patients with atherosclerosis obliterans when compared with 105 age-matched controls.63 As opposed to findings in CAD, miR-21 presence was significantly higher in sera of sclerotic patients, while levels of miRs -221 and -222 were unchanged when compared with controls. The presence of circulating microRNAs in atherosclerosis was also studied selectively in HDL particles, whose microRNA content was found to change in subjects with familial hypercholesterolaemia, a genetic disorder of the LDL receptor which results in extreme atherosclerosis in homozygous cases.53 In HDL of healthy subjects, miR-223 was in the top 10 of highest expressed microRNAs, but its levels increased dramatically in HDL of familial hypercholesterolaemia subjects. Also in atherosclerotic apolipoprotein E and LDL receptor knockout mice, HDL particles were found to contain increased amounts of miR-223 when compared with controls. No other prominent inflammatory microRNAs, such as miRs -21, -221, -155, and 146a/b, stood out, only miR-222 seemed increased in HDL of familial hypercholesterolaemia, similar to miR-223 but less dramatic. A preliminary conclusion from these early studies could be that the circulating microRNA repertoire of CAD and atherosclerosis surprisingly diverges.

5.3 Circulating microRNA profiles of diabetes

Circulating microRNAs have also been proposed as novel biomarkers for diabetes.60,64,65 In an early study, Chen et al.64 studied not only sera of subjects with DM but also of subjects with non-small cell lung carcinoma and compared them to healthy controls. Interestingly, they found that there was considerable overlap in serum microRNA presence for the two diseases and not for controls, and attributed this to the general inflammatory response seen in these diseases. Although these authors state that the overlapping microRNAs may be related to the body's immune system, unfortunately there is no detailed information on their identity.

Zampetaki et al.60 screened pools of diabetic sera derived from the Bruneck cohort for the expression of 754 microRNAs, detected 130 of them and found 30 microRNAs to be differentially present between diabetic and control pools. Thirteen topologically unique microRNAs were selected for further validation in a larger group of 80 DM patients vs. 80 matched controls. Here, most miRs decreased significantly in DM, including miR-126, -21, and -223. Interestingly, levels of some miRs including -126 and -223 were already altered before DM manifestation. MiR-126 presence was analysed in the entire Bruneck cohort of 822 subjects, and emerged as a significant predictor of manifest DM and even correlated negatively with increasing glucose intolerance. These authors propose the use of the five top-ranked microRNAs, miR-15a, -29b, -126, -223, and -150, as highly sensitive biomarkers of DM.

Recently, serum levels of seven microRNAs, selected for their proven involvement in diabetes-related processes, were studied in small groups of patients with established DM type II when compared with patients who either were susceptible (with comparably high BMIs of just above 26) or had pre-diabetes.65 Here, all seven studied microRNAs were found increased in diabetic subjects, including miRs -9, -34a, and 146a, which are linked to inflammation and/or ageing (Tables 1 and 2). None of these seven was identified by Zampetaki et al.60 as circulating biomarkers of DM. While Zampetaki et al.60 predominantly found down-regulated microRNAs in diabetic sera, with one exception (miR-28-3p), the seven studied microRNAs were all up-regulated in sera of DM patients. Finally, Caporali et al.66 found increased levels of the EC miR-503 in sera of diabetic individuals, and suggested this microRNA to have a role in angiogenesis.

View this table:
Table 1

MicroRNAs in TLR signalling: involvement in HF

MicroRNARegulation in HFRole in HFReferenceRole in human inflammatory autoimmune diseases (Dai and Ahmed84)
let-7Inhibits cardiac hypertrophy66
miR-9Targets myocardin to inhibit hypertrophy67
miR-16↑/↓68,69,70,71RA
miR-21Controls extend of interstitial fibrosis and cardiac hypertrophy15SLE
miR-27bUp-regulated early in hypertrophy, role in angiogenesis85
miR-10686
miR-125b86SLE (125a)
miR-132Targets cardiac L-type Ca channel β2 subunit protein69,71,73,74RA
miR-145Up-regulated in human aortic stenosis86
miR-146Doxorubicin-induced HF8SLE, RA
miR-155Targets AT1R12,13RA, MS
miR-199Regulates cardiac myocyte hypertrophy7577
miR-221↑/↓86
miR-223Diabetic HF10SLE, RA
miR-466l86
  • Based on Table 2 in O'Neill et al.5 MicroRNAs highlighted in bold are also involved in ageing. MiR-105, -348, -579, and -369-3p are also part of the TLR signalling pathway but have no proven regulation in HF. SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; MS, multiple sclerosis.

View this table:
Table 2

MicroRNAs in ageing pathways: involvement in HF

MicroRNARegulation in HFRole in HFReferenceRole in human inflammatory autoimmune diseases (Dai and Ahmed84)
let-7b86
miR-1Anti-hypertrophic86
miR-21Controls extend of interstitial fibrosis and cardiac hypertrophy15SLE
miR-24Suppresses cardiomyocyte and EC apoptosis78,79
miR34aRole in endothelial senescence, predisposing to atherosclerosis58,73MS
miR-100Involved in the beta-adrenergic receptor-mediated repression of ‘adult’ cardiac genes87
miR-10686
miR-12886
miR-132Targets cardiac L-type Ca channel β2 subunit protein69,71,73,74RA
miR-138Modulates cardiac patterning during embryonic development81
miR-14086
miR-145Up-regulated in human aortic stenosis86
miR-146Doxorubicin-induced HF8SLE, RA
miR-199Regulates cardiac myocyte hypertrophy7577
miR-206Associated with protection against cardiac remodelling after MI 88
Contributes to high glucose-mediated apoptosis in cardiomyocytes83
miR-21786
miR-302-36786
miR-32086
miR-499Causes cellular hypertrophy and cardiac dysfunction89
(/↑)Inhibits cardiomyocyte apoptosis85
  • Based on Table 1 in Chen et al.6 MicroRNAs highlighted in bold are also involved in TLR signalling. SLE, systemic lupus erythematosus; RA, rheumatoid arthritis; MS, multiple sclerosis.

Recently, haematopoietic cell-derived microRNAs, such as the above-mentioned miRs -146a, -155 and -223, were shown to confound circulating microRNA levels.67 Also miRs -21, -221, -222, and -423-5p are among the circulating microRNAs that can be of haematopoietic cell origin. Remarkably, miRs -21, -146a, and -223 are often among the top-detected microRNAs in serum profiling studies.53,58,60 This warrants the cautious interpretation of circulating inflammation-related microRNA levels. Indeed, as mentioned above, the agreement between different studies on vascular disease is poor, and the field needs larger patient group studies with standardized measurement methods to establish the exact presence, including identity and origin, of microRNAs in the circulation.

5.4 Functional roles for circulating microRNAs

That circulating microRNAs do more than just being available as disease biomarkers is becoming increasingly clear. Indeed, HDL-bound microRNAs are delivered to recipient cells and were also shown to modulate the target gene expression in these cells.53 Microvesicles also use microRNAs to mediate intercellular communication, and microRNAs from lung were shown to end up in bone marrow cells in culture and actively repress production of pulmonary epithelial cell mRNAs in these marrow cells.68 The same principle would hold true for cardiac tissue-derived microvesicles, and is presumably a mode of communication between systemic organs and the core of the immune system.68 Vice versa, microparticles secreted from mesenchymal stem cells were readily taken up by H9C2 cardiac myocytes.69

On a local tissue level, adipocytes were found to communicate to each other using microvesicles, and vesicles released from large adipocyte-stimulated lipid storage in smaller adipocytes using microRNAs as messengers.70 In addition, two independent studies showed inter-communication of ECs using miR-126.60,71 Endothelial apoptotic bodies were shown to contain microRNAs and high glucose lowered their miR-126 content.60 MiR-126 was also found as part of microparticles that are produced by apoptotic ECs during atherosclerosis.71 Importantly, these microparticles conveyed a survival signal to neighbouring ECs via miR-126 and its target, regulator of G-protein signalling 16, a G protein coupled receptor inhibitor, allowing the production of CXCL12 and thereby antagonizing apoptosis. These studies uniquely show a paracrine signalling function for microRNAs during the process of atherosclerosis.

It is still too early to speculate on whether a distinction can be made between non-cell-free microRNAs—that is, microRNAs encapsulated in transport vesicles—and cell-free microRNAs in terms of functionality and possible ‘second messenger’ roles.

6. Ageing, inflammation, and HF: are there shared microRNAs?

It is unclear whether inflammation and cardiovascular morbidity converge into the process of ageing, or vice versa, whether ageing accelerates inflammation and cardiovascular diseases. Below, we tried to link these together and find common microRNA pathways. Since inflammation is rather a broad concept, we focused on TLR signalling, which is central in HF, the development of the metabolic syndrome and ageing.7274 A recent review lists microRNAs involved in all levels of this central pathway in inflammation,7 and it is intriguing that most of these microRNAs have proven regulation and/or function in HF (Table 1). In addition, most of these microRNAs seem to be up-regulated in failing hearts. The same holds true for microRNAs involved in pathways that modulate the ageing process, recently reviewed,6 where we see that all microRNAs listed are also involved in HF (Table 2). In addition, there is some overlap between microRNAs involved in inflammation and ageing (almost 50%, as highlighted in both tables). Therefore, HF-associated microRNAs appear to have central roles in both inflammation and ageing.

Our group was the first to study microRNAs in ageing-associated HF, and identified miR-18 and -19 to be down-regulated in failure-prone-aged mice and in cardiac biopsies of HF patients of age.5 This down-regulation was linked to increased expression of the matricellular proteins thrombospondin-1 and connective tissue growth factor, and to increased fibrosis in aged failing myocardium. Interestingly, mice on a failure-protected genetic background showed inverse expression patterns of both these microRNAs and matricellular proteins, with miRs -18 and -19 being up-regulated with ageing, indicating the possibility of their contribution to healthy ageing.

MicroRNAs -155, -21, and -146 have established roles in immune and inflammatory pathways, and are central in TLR signalling.75 MiR-21 has been shown to target the tumour suppressor programmed cell death protein 4 (PDCD4), a proinflammatory protein that promotes activation of the transcription factor NF-κB and suppresses IL-10.76 By targeting PDCD4, miR-21 becomes a negative regulator of TLR4 signalling.76 In addition, miR-21 has been shown to be induced by STAT377 and NF-κB,76 central transcription factors in immune functions but also in cardiac hypertrophy.

EC senescence is also thought to play a role in cardiovascular diseases such as atherosclerosis.1,4 During endothelial senescence, microRNAs -34a and -217 were found up-regulated.78,79 These endothelial microRNAs triggered endothelial senescence in part through Sirtuin 1, a class III histone deacetylase which is linked to ageing and to the regulation of the level of inflammatory responses.80 MiR-34a was also shown to be up-regulated in the heart and spleen of older mice.78 In addition, miR-20c was induced by oxidative stress in ECs, and its over-expression induced EC growth arrest, apoptosis, and senescence via zinc finger E-box binding factor 1.81

MiR-146a has roles in autoimmunity, and mice lacking this gene show accelerated ageing.36 From 6 to 8 months of age, miR-146a-deficient mice, which are on an ageing-susceptible genetic background of 129/Bl6,5 exhibit signs of immunoproliferative disease, characterized by splenomegaly, lymphadenopathy, and premature death. Liver, kidneys, and lungs had lymphocytic and monocytic infiltrates with some evidence of tissue damage. Recently, miR-146a was found to decrease with ageing of ECs, where it targeted NADPH oxidase 4, involved in cell senescence and ageing.82 In agreement with this, miR-146a decreased in models of organismal ageing, including foreskin and CD8+ T cells of old vs. young donors.83 Another microRNA with a role in inflammation, miR-221, increased with ageing in ECs.82 However, this miR decreased in organismal ageing.83 In addition, miR-155 presence in sera of 53 healthy and CAD patients showed a significant negative correlation with age.59

7. Conclusions and future perspectives

MicroRNAs have established roles in all aspects of organismal development and disease. In recent years, their roles in inflammation, ageing, cancer, and cardiovascular diseases are proven beyond doubt. As reviewed here, microRNAs play major roles in the inflammatory aspects of vascular disease. Given the central role of inflammation in HF, with inflammatory transcription factors including NF-κB and STAT3 and with inflammatory cytokines including TNFα and IL-6 as central mediators of cardiac hypertrophy, a role for inflammatory microRNAs in the development of HF seems ensured but still needs to be proven.

Whether microRNAs will be used some day for the diagnosis of inflammatory involvement in cardiovascular and/or other inflammatory diseases depends on the precise characterization of the role and distribution of microRNAs in the blood. Inflammation-associated microRNAs such as miR-21, -146, and -223 are always detected at high levels in the blood, also in healthy subjects, and to date it is unclear what their function is. Their presence might even be the result of leakage from blood cells, in which these microRNAs have established high levels. Therefore, it is of great importance that standardized techniques are developed for the detection of circulating microRNAs.

Some circulating microRNAs, apparently those contained in some sort of vesicle, appear to function as messengers between cells. This may be a way of tissues including the heart and vessels, to communicate internally but also with the immune system, possibly changing each other's destiny. In conclusion, the potential of microRNAs for the diagnosis and treatment of human disease seems to be ever-growing, and with the discovery of other non-coding RNAs, the field of non-coding RNAs will keep on expanding for the coming years.

Funding

B.S. received a Veni grant (016.096.126) from the Netherlands Organization for Scientific Research, a Horizon grant (93519017) from the Netherlands Genomics Initiative, and a research grant from the Netherlands Heart Foundation (NHS 2009B025). S.H. received a Vidi grant from the Netherlands Organization for Scientific Research (91796338) and research grants from the Netherlands Heart Foundation (NHS 2007B036 and 2008B011), Research Foundation – Flanders (FWO 1183211N, 1167610N, G074009N), European Union, FP7-HEALTH-2010, MEDIA, Large scale integrating project, and European Union, FP7-HEALTH-2011, EU-Mascara, Large scale integrating project.

Acknowledgements

We thank the colleagues at the Center for Heart Failure Research for fruitful discussions.

Conflict of interest: none declared.

Footnotes

  • This article is part of the Review Focus on: The Role of MicroRNA in Cardiovascular Biology and Disease

References

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