MicroRNA Signature and Cardiovascular Dysfunction

Arunachalam, Gnanapragasam PhD*; Upadhyay, Rohit PhD*; Ding, Hong MD, PhD*,†; Triggle, Chris R. PhD*,†

Journal of Cardiovascular Pharmacology: May 2015 - Volume 65 - Issue 5 - p 419–429
doi: 10.1097/FJC.0000000000000178
Highlighted Meetings Article

Abstract: The worldwide increase in the prevalence of obesity and type 2 diabetes and the associated elevated risk of cardiovascular disease (CVD) has emphasized the need to seek new therapeutic targets to offset the negative impact on human health outcomes. In this regards, microRNAs (miRNAs), a class of small noncoding RNAs that mediate posttranscriptional gene silencing, have received considerable interest. miRNAs repress gene expression by their ability to pair with target sequences in the 3′ untranslated region of the messenger RNA. miRNAs play a crucial role in the biogenesis and function of the cardiovascular system and are implicated as dynamic regulators of cardiac and vascular signaling and pathophysiology. Numerous miRNAs have been identified as novel biomarkers and potential therapeutic targets for CVD. In this review, we discuss the contribution of miRNAs to the regulation of CVD, their role in macrovascular/microvascular (dys)function, their potential as important biomarkers for the early detection of CVD, and, finally, as therapeutic targets.

Departments of *Pharmacology; and

Medical Education, Weill Cornell Medical College in Qatar, Doha, Qatar.

Reprints: Chris R. Triggle, PhD, Departments of Pharmacology and Medical Education, Weill Cornell Medical College in Qatar, Education City, Doha 24144, Qatar (e-mail: cht2011@qatar-med.cornell.edu).

Supported by Qatar National Research Foundation, National Priorities Research Program Grants to C. R. Triggle and H. Ding (4-910-3-244, 5-149-3-040 and 6-428-3-113).

The authors report no conflicts of interest.

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivatives 3.0 License, where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially.

Received August 29, 2014

Accepted October 15, 2014

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Despite advances in our understanding of the etiology, cellular and molecular mechanisms that contribute to cardiovascular disease (CVD), the morbidity and mortality associated with CVD remains a serious concern for developed and developing countries.1 Comorbid conditions, such as obesity, diabetes, hypertension, and dyslipidemia, have been extensively studied and identified as major risk factors for CVD.2,3 Although a wide array of prognostic, diagnostic, and therapeutic tools are available, the prevalence continues to increase, stressing the need for further insight into the molecular mechanisms that contribute to the pathology of CVD and searching for new innovative therapeutic strategies.

Mutations and/or aberrant expression of vascular genes and their regulation through small noncoding RNAs have emerged as crucial events in vascular signaling that contribute to the progression of CVD.3–7 Small noncoding RNAs, called microRNAs (miRNAs or miRs) that function as important posttranscriptional regulators have been implicated in a broad range of vascular function, including cell differentiation, proliferation, survival, metabolism, and development,8 and therefore are regarded as intriguing potential tools for the detection and treatment of CVD.9–11 This review focuses on the putative/potential role of the numerous miRNAs that have been identified as important regulators of cardiovascular function and contributors to cardiac and macrovascular/microvascular dysfunction and addresses the potential of miRNAs as biomarkers and therapeutic targets for the treatment of CVD. As will be evident, there are many questions that still need to be addressed and differences in conclusions reached by different studies may reflect differences in protocol design, species differences, or those related to acute versus chronic stages of the disease.

miRNAs represent approximately 4% of the genes in the human genome with several thousand sequences identified. miRNAs were originally discovered in Caenorhabditis elegans by Victor Ambros and Gary Ruvkun in 199312; however, their role was not recognized for about another 7 years when a series of publications appeared.13 The first miRNA database (release 1) was announced in December 2002 having only 218 entries, but the 2014 database (release 21; June 2014) has 28,645 miRNA entries. The latest miRBase Sequence Database contains 28,645 entries representing hairpin precursor miRNAs, expressing 35,828 mature miRNA products, in 223 species. With the database release, 20,4196 new hairpin sequences and 5441 new mature products have been added, including the first miRNAs in 17 species.14 These statistics indicate how rapidly this field is expanding. A challenge for the further development of miRNAs as therapeutic targets is that a single miRNA can regulate several, if not hundreds of, targets. In addition, the presence of RNases in the extracellular milieu may reduce the stability of exogenously administered miRNAs. Nevertheless, enhancing our knowledge of the miRNAs that target specific proteins of cell signaling pathways will aid us in unraveling the pathogenesis of CVD.

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miRNAs are a large family of posttranscriptional regulators, comprising approximately 22 (15–27) nucleotides and small, noncoding single-stranded RNAs that are derived from endogenous hairpin-shaped transcripts and have emerged as key posttranscriptional modulators of diverse gene expression in biological machinery. miRNAs regulate gene function at the posttranscriptional level by messenger RNA (mRNA) degradation, translational repression, or miRNA-mediated mRNA decay.

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The multistep biological process involves (1) transcription, (2) processing, (3) splicing and export, and (4) maturation and target binding and are summarized in Figure 1. miRNA biogenesis begins with RNA polymerase II–mediated transcription of miRNA (termed pri-miRNA) gene from either an independent miRNA gene or portions of introns of protein coding RNA polymerase II transcripts. A single pri-miRNA often contains the sequence for several miRNAs, which is then subsequently cleaved by the RNA-specific RNase-III–type endonuclease Drosha and its cofactor DGCR8 into ∼70-nucleotide precursor miRNAs (called pre-miRNAs). The pre-miRNAs formed in the nucleus are transported to cytoplasm through nuclear pores by exportin-5 and RanGTP-binding protein.15–18

In the cytoplasm, the pre-miRNA is subjected to enzymatic processing and cleaved into a ∼22-nucleotide miRNA duplex by the cytoplasmic RNase-III endonuclease enzyme Dicer that is coded by the DICER gene and its cofactors, such as trans-activation RNA-binding protein, TREP (Fig. 1). The double-stranded miRNA duplex is typically composed of a mature guide strand (functional and active miRNA) and passenger strand (inactive miRNA*). Following target binding, unwinding of duplex, the active strand is loaded into RNA-induced silencing complex called, RISC, which is a multi-protein complex that consists of Argonaute family proteins and other regulatory effectors mediating the inhibitory action of RISC. Finally, the RISC directs the mature miRNA strand to bind to target mRNA through complementary binding of their seed sequence (miRNA 5′ end, ∼6  nucleotides, which is complementary to 3′ untranslated region of mRNA), thus facilitating gene silencing or degradation.18–21

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Regulation of MicroRNA Gene Transcription

miRNAs regulate their own biogenesis through single- or double-negative feedback loops with specific transcription and cofactors. Indeed, the nuclear cleavage can be delayed and inhibited by interaction with transcription factors, such as SMADs and estrogen receptor-α, which can modulate the catalytic activity of Drosha. Additional factors, including several RNA-binding proteins and systemic and dynamic activities of cell environment, can also regulate miRNA transcription.17,22,23

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Important developmental, functional, and pathological roles for miRNAs have all been described in the regulation of vascular genes that play an extensive role in vasculogenesis and vascular remodeling.9,24–26

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MicroRNAs as Dynamic Modulators of Cardiovascular Development

The vital role of miRNAs during cardiovascular development has been well-described using tissue- or cell-specific deletion/mutation of genes that encode key enzymes/proteins/cofactors involved in miRNAs biogenesis.6,24,27,28 Selective silencing of Dicer enzymes in mice during early stages of embryogenesis (heart development) and in postnatal cardiomyocytes has resulted in embryonic lethality and severe cardiac phenotypes, such as hypertrophy, fibrotic lesions, dilated cardiomyopathy, and heart failure.29–32 Notably, Dicer mutation during embryonic stage results in defective blood vessel formation in embryos and yolk sacs, with aberrant expression of vascular endothelial growth factor (VEGF) and its receptors 1 and 2 (VEGFR-1 and VEGFR-2).33

Sequence- and array-based miRNA expression profiling studies indicate that several distinct families of miRNAs are involved in cardiac development and function. miR-1 and miR-133, the 2 abundant miRNAs in heart, have been reported to have differential roles in heart, with miR-1 promoting cardiomyocyte proliferation while miR-133 inhibiting proliferation.6,26,34 Conversely, miR-27 has a modulating role during early cardiogenesis35 and target deletion of miR-1-2 has been associated with cardiac arrhythmias and sudden cardiac death.36 In addition, miR-208a, miR-208b, miR-499, miR-126,6,27,37 and members of the miR-15 family (ie, miR-15a/b, miR-16-1/2, miR-497, and miR-195) have been reported to play important regulatory roles during cardiac development, integrity, and regulate proliferation, premature cell cycle arrest, and cardiac ischemic injury.38,39 Upregulation of the miR-17-92 cluster directly downregulates the cardiac progenitor genes Isl1 and Tbx1, thereby facilitating myocardial differentiation.40 Knockdown of the miR-145/miR-143 cluster, which is enriched in vascular smooth muscle cells (VSMCs), results in enhanced proliferation and migration of VSMCs.41,42 Other miRNAs including let-7f, miR-130, and miR-210 have also been reported to play contributing role during blood vessel formation and tubulogenesis.27

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MicroRNAs and Cardiovascular Disease

MicroRNAs in Heart Failure

The diverse role of several miRNAs in heart failure has been extensively investigated using genetic deletion protocols in mice and cell culture models.43 The cardiomyocyte-specific miR-1 is a potential target in heart failure, and SERCA2 gene therapy restores miR-1 expression in a chronic post-MI rat model of heart failure.44 In addition, adenovirus targeted delivery of miR-1 reverses pressure-overload cardiac hypertrophy.45 However, upregulation of miR-1 also occurs in ischemic myocardium in acute myocardial infarction, with reversal following treatment with propranolol, and elevated circulating miR-1 is present in patients with acute MI.46–49 Furthermore, right atrial biopsies from patients undergoing coronary artery bypass grafting illustrated a downregulation of miR-133a, but not miR-1.50 Potential species (rat vs. human) and/or differences between acute versus chronic post-MI may explain these discrepancies regarding the role of miR-1.

Downregulation of fibroblast-rich miR-29 alters the expression of extracellular matrix genes (fibrillins and elastin) and accelerates fibrosis. In contrast, downregulation of miR-29 suppresses cardiomyocyte apoptosis.51,52 miR-15 is upregulated in infarcted cardiac tissue and endogenously silencing miR-15 in cardiomyocytes decreases cardiomyocyte apoptosis.39 miR-24 overexpression in cardiomyocyte triggers myocardial healing by attenuation of fibrosis and apoptosis; however, miR-24 silencing in endothelial cells (ECs) has an opposite effect as apoptosis of ECs is inhibited.53–55 These data stress the difficulties in the therapeutic targeting of a single miRNA. Adenoviral-mediated delivery of miR-101a to the infarcted heart has beneficial effects on cardiac function by reducing the development of fibrotic scar tissue.56 Knockdown of miR-214 in an ischemia-reperfusion injury mouse model triggers cardiomyocyte apoptosis and fibrosis.57 Under ischemic stress, the overexpression of miR-499 in the heart reduces apoptosis and infarct size, whereas knockdown of miR-499 increases myocardial apoptosis and infarct size.58 Boon et al59 reported that miR-34a levels are augmented in aged mice heart and its inhibition reduces age-associated cardiomyocyte cell death.

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Cardiac Hypertrophy

There is an expanding database indicating that miRNAs are involved in the pathogenesis of cardiac hypertrophy, and miRNAs are differentially and temporally regulated in both physiological (normal) and pathological (deleterious) hypertrophy growth.60 For instance, in addition to its developmental role, the functional loss of miR-1 depresses its downstream effectors, such as MEF2A, Twif1, and IGF-1, and increases cardiac mass and wall thickness.60–63 The genetic deficiency of miR-133a-1 and miR-133a-2 (bi-cistronic cluster of miR-1) results in extensive hypertrophic growth associated with an upregulation of calcineurin and reflects a reciprocal repression between miR-133 and calcineurin.64,65 Adenoviral-mediated modulation of miR-26 affects myocyte survival and hypertrophy by targeting of the transcription factor Gata4.60 Under pro-hypertrophic conditions, a miR-9 mimic decreases expression of myocardin and inhibits cardiac hypertrophy.66 Upregulation of miR-98 in mice modulates Trx1 and cyclin D2 and reduces angiotensin II–induced cardiac hypertrophy.67 Several pro-hypertrophic miRNAs have been identified; however, their individual roles in the regulation cardiac hypertrophy remains to be clarified.60 Overexpression of miR-195 induces hypertrophy in cultured neonatal cardiomyocyte and dilated cardiomyopathy in vivo.68 Furthermore, the myomiRNA families of miR-208a, miR-208b, and miR-499 are located within the myosin heavy chains genes Myh6, Myh7, and Myh7b, respectively, and play important roles in response to hypertrophic stress.37 In contrast, upregulation of miR-208a activates Myh7 and induces cardiac hypertrophy, resulting in cardiac systolic dysfunction.69 During ischemic conditions, miR-499 suppresses hypertrophy-mediated apoptosis and myocardial infarction.58 Similarly, upregulation of miR-199a-5p in hypertrophic heart has been reported to downregulate the transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) and expression of the histone deacetylase, sirtuin (SIRT1), whereas knockdown of miR-199a decreases cardiomyocyte size and attenuates isoproterenol-induced hypertrophy.70,71 Data from in vitro and in vivo studies indicate a controversial role for miR-18b, which is increased in cardiac hypertrophy; however, inhibition of endogenous miR-18b induces hypertrophic growth.60,72 Other cardiac enriched miRNAs that are critically implicated in the development of cardiac fibrosis (reviewed elsewhere73,74) and other cardiac regenerative strategies, including cardiomyocyte proliferation, differentiation, survival, and reprogramming, are summarized in Figure 2.

Although it is clear that miRNAs play an important role in the regulation of cardiac function, however, the question remains: “Is there one specific, or at least a limited number of, miRNA(s) that can serve as a target, or biomarker, for heart failure, cardiac hypertrophy and fibrosis in patients?” In this regard, Roncarati et al75 report changes in the composition of 12 miRNAs, miR-27a, -199a-5p, -26a, -145, -133a, -143, -199a-3p, -126-3p, -29a, -155, -30a, and -21, that were significantly increased in the plasma of patients with hypertrophic cardiomyopathy with miR-199a-5p, -27a, and -29a correlated with hypertrophy, but only an elevation of miR-29a correlated with both hypertrophy and fibrosis. However, only 41 patients were included in this study and additional studies are required to extend and confirm these findings. In another analysis of circulating miRNAs in heart failure patients, elevated miR-29a was noted and potentially linked to its known antiproliferative function and therefore a link to the reduced self-repair capacity of the adult heart.76

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MicroRNAs in Vascular Disease

Both ECs and VSMCs play key roles in determining vascular integrity and function and, as depicted in Figure 3, aberrant expression of miRNAs is linked to vascular pathologies, including atherosclerosis, coronary artery disease, vascular inflammation, and diabetic vascular complications.

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MicroRNAs Involved in Endothelial Physiology: Role in Endothelial Cell Senescence

EC senescence is defined as premature “permanent cell cycle arrest” and is characterized by apoptosis, inflammation with decreased production of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS), and resultant endothelial dysfunction, followed by atherosclerosis and accelerated aging.77,78 The contribution of miRNAs in the aging process of ECs has been reviewed.79,80 Hearts of aged mice have an increase in miR-34a expression when compared with hearts from young mice.81 The miR-34a expression is increased during senescence and cell cycle arrest and overexpression of miR-34a accelerates senescence through inhibition of SIRT1 in ECs.81 In addition, overexpression of miR-34a in endothelial progenitor cells (EPCs) results in an increase in EPC senescence with impaired SIRT1 expression and angiogenesis.82 Similarly, increased levels of miR-217 and also mi34a result in the downregulation of several important targets of SIRT1, including FoxO1 and eNOS, thus promoting premature EC senescence and apoptosis.82,83 Several other studies have also demonstrated that increases in the expression of miR-200c and miR-146a are associated with aging, with the latter directly targeting the NADPH subunit, nox4, modulate growth arrest, senescence, and apoptosis in ECs.84,85

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Role in Angiogenesis

Loss or impaired function of ECs function is a characteristic feature of impaired angiogenesis and a key contributor to increased inflammation and the development of ischemic heart disease, vascular disease, inflammation, and atherosclerosis. A group of miRNAs, referred to as angiomiRNAs, are important in the regulation of angiogenic processes and responses.86,87 miR-126 is enriched in ECs and is recognized as a master regulator of vascular integrity and angiogenesis. miR-126−/− mice usually die during embryonic development as a result of ruptured blood vessels and mice that survive into adulthood display defective cardiac neovascularization.88,89 Induction of miR-126 is involved in pro-angiogenesis by enhancing VEGF and angiopoietin 1 signaling through inhibition of Sprouty-related protein (SPRED1) and phosphoinositol-3 kinase regulatory subunit 2 (PIK3R2/p85-beta) expression that are essential for the maintenance of vascular integrity.89–91 Moreover, based on the measurement of plasma and EPC-associated miRNAs in patients, the downregulation of miR-126 and subsequent induction of SPRED1 gene activity has been associated with diabetes.92,93 Thus, reduced miR-126 expression in EPCs may partially be responsible for impaired vascular repair in diabetic patients.93 Moreover, transfection with anti-miR-126 blunts proliferation and migration of EPCs and comparable reductions in miR-126 and other miRNAs have been observed in hyperglycemic ob/ob mice.92 Overexpression of miR-221/miR-222 inhibits tube formation, migration, and wound healing of human umbilical vein endothelial cells (HUVECs) in vitro by modulating its downstream target, c-kit (a marker of EPCs) expression, the receptor for cardiac stem cell factor, without affecting mRNA expression, thus suggesting posttranscriptional regulation.94 miR-221/miR-222 overexpression also indirectly reduces the expression of eNOS in Dicer siRNA-transfected cells.95 Evidence for the anti-angiogenic function of miR-221/miR-222 cluster is provided by its role in the control of the expression of signal transducer and activator 5 (STAT 5), which normally promotes vessel formation and maturation.96 Furthermore, miR-221 downregulates ZEB2, thus enhancing p21 expression; as a result, EC proliferation is decreased and cell cycle arrest is promoted.97 miR-17-92 is a polycistronic cluster, functionally categorized into 4 families: miR-17, miR-18, miR-19, and miR-92, with the cluster playing a complex role in the overall regulation of angiogenesis.80 miR-92a, the most prominent member in this cluster, decreases the expression of integrin subunit α5 and SIRT1 and thereby suppresses angiogenesis of ECs.98 In contrast, both miR-20a and miR-92a are reported to display anti-angiogenic activity by negatively targeting several pro-angiogenic proteins, including VEGF-A, integrin subunit α5 (ITGα5), and integrin β5 (ITGB5) expression.98 Overexpression of miR-17, miR-18a, miR-19a, and miR-20a inhibits endothelial sprouting in vitro while inhibition of miR-17 and miR-20a increases the number of lectin-perfused vessels in matrigel plugs.99 These findings indicate distinct functions of this family of miRNAs in the regulation of angiogenesis.

The miR-23-27-24 cluster is abundant in ECs. In mice, miR-23 and miR-27 expression is essential for promoting pathological angiogenic signaling through Sprouty2 and expression of the transmembrane semaphorin protein Sema6A.100 Changes in the expression of miR-24 are linked to the induction of EC apoptosis and inhibition of cell sprouting and migration.53 However, another study has reported that miR-24 expression inhibits cardiomyocyte apoptosis in both an in vitro protocol and an in vivo mouse model of acute myocardial infarction. This effect is, at least in part, mediated by direct repression of BH3-only domain-containing protein (Bim).55 miR-27 expression has been shown to play a crucial role in angiogenesis by targeting of thrombospondin-1 and enhancing atherosclerotic plaque maturation.100 miR-16 and miR-424 are members of the family of anti-angiogenic miRNAs and blunt the expression of VEGFR-2, FGFR1, and VEGF in ECs and the overexpression of either miR-16 or miR-424 reduces proliferation and migration and impairs EC function.101 miR-101102 and miR-200b103 are also anti-angiogenic and, when inhibited, result in the promotion of angiogenesis. miR-130a104 is pro-angiogenic and reduces the expression of anti-angiogenic genes, GAX and HOXA5. miR-296105 and miR-132106 are also pro-angiogenic and increase EC proliferation and branching. In addition, miR-210107 functions as an angiogenic switch and promotes EC migration and tube formation by suppressing the expression of the receptor protein-tyrosine kinase, Ephrin-A3.

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Role in Vascular Inflammation

Vascular inflammation is an integral and early event in atherogenesis, characterized by leukocyte activation/infiltration and release of inflammatory cytokines and cell adhesion molecules. Vascular inflammation results in changes in the expression, both induction and inhibition, of many miRNAs in ECs. In addition to its role in angiogenesis,89 miR-126 also downregulates tumor necrosis factor α (TNF-α)–induced expression of the vascular cell adhesion molecule-1 (VCAM-1), which mediates leukocyte adherence to ECs.108 miR-21 is also a positive regulator of neointima lesion formation and downregulation of miR-21 significantly decreases neointima formation in balloon-injured rat carotid arteries.109 The targets for miR-21 in mediating its effects on VSMC proliferation and apoptosis are the tumor suppressor phosphatase, phosphatase, and tensin homolog (PTEN) that serves to inhibit the Akt “cell survival” pathway and the anti-apoptotic protein, Bcl-2.109 The problem of selective manipulation of miRNAs to address vascular dysfunction has been addressed in a study designed to inhibit restenosis, but, at the same time, preserve endothelial function.110 As already noted, miR-126 is highly expressed in ECs and plays a critical role in angiogenesis and reduced expression is associated with a loss of vascular integrity.89–91 miR-126 represses the expression of the negative regulators of the MAPK and PI3K pathways.89 The problem of restenosis has been addressed in a study by Santulli et al,110 wherein an adenoviral vector was designed that incorporated the cell cycle inhibitor p27 and target sequences for miR-126 such that overexpression occurred in VSMCs but not ECs. The proof-of-principle study by Santulli et al110 demonstrates that it is possible to selectively target VSMC proliferation without affecting EC function and therefore, potentially, provides a novel therapeutic approach to prevent angioplasty–restenosis and related thrombosis.

miR-181b inhibits importin-3 expression, a protein required for nuclear translocation and activation of NF-κB, thus the overexpression or systemic administration of miR-181b results in a block of NF-κB–mediated induction of VACM-1 expression and reduces the leukocyte accumulation and activation of ECs.111 Fang et al112 have demonstrated that miR-10a expression was reduced in atherosusceptible regions of inner aortic arch and aorta–renal branches of swine and expression of monocyte chemoattractant protein 1 (MCP-1), interleukin 6 (IL-6), IL-8, VCAM-1, and E-selectin is increased, thus predisposing the blood vessel to inflammation. Furthermore, knockdown of miR-10a decreases the expression of the mitogen-activated kinase kinase kinase, MAP3K7, and β-transducin repeat-containing gene, beta TRC, the key regulators of NF-κB activation, therefore implicating that the regulatory role of miR-10a in the inflammatory response in ECs is through the NF-κB pathway.112 In HUVECs, TNF-α induces miR-31/miR-17 expression, resulting in a negative feedback that limits the development of inflammation by regulating E-selectin and miR-17-3p expression and controlling the release of ICAM-1 enhanced neutrophil adhesion.113 miR-155 targets Ets-1, which is a key transcription factor stimulated by angiotensin II, and TNF-α and thrombin expression, thereby playing a key role in inflammation, including the regulation of VCAM1 and monocyte chemotactic protein 1 (MCP1).114,115 Likewise, several miRNAs, including miR-150, miR-17-92 (regulate B-cell differentiation), as well as miR-424, miR-17-5p-20a-106a, and miR-146 (regulating monocyte/macrophage differentiation), have also been implicated as playing a role in modulating vascular inflammation and remodelling.116

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Diabetic complications are predominantly of vascular origin and typically classified as microvascular (nephropathy, retinopathy, microangiopathy) or macrovascular disease including CVD.

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MicroRNAs and Diabetes-associated Endothelial Dysfunction

Diabetes-associated vascular phenotypes most often begin with endothelial dysfunction and impaired angiogenesis. In myocardial microvascular ECs from diabetic Goto-Kakizaki rats, elevated levels of miR-320 are associated with diminished cell proliferation, migration, and a lower expression of insulin-like growth factor, IGF-1, that can all be reversed by transfection with a miRNA-320 inhibitor.117 Exposure of HUVECs to high glucose (HG) in culture increases expression of miR-221 with reduced c-kit expression, whereas miR-222 silencing attenuates HG-mediated c-kit repression.118 In another study, through gain and loss of function protocols, miR-221/miR-222-mediated posttranscriptional regulation of P27KIP1 and P57KIP2 has been shown to be crucial for HG-mediated and advanced glycation end product, AGE-mediated vascular cell damage.119 miR-200c is also upregulated in ECs by oxidative stress and thus also likely linked to hyperglycemia-induced oxidative stress and endothelial dysfunction.84 Aortic VSMCs isolated from db/db diabetic mice showed increased miR-125b expression along with downregulation of Suv39h1, a histone–lysine N-methyltransferase.120 This increase in miR-125b is linked to increased expression of inflammatory proteins, IL-6, and monocyte chemotactic protein 1 and increased monocyte–VSMCs binding in hyperglycemic conditions, thus indicating a role for miR-125b in accelerating atherosclerosis.120 Under hyperglycemic (25 mM of glucose) cell culture conditions and ischemic starvation of ECs (HUVECs and human microvascular ECs), an increase in miR-503 expression results in reduced proliferation, migration, and network formation.121 In addition, miR-503 expression was markedly higher in calf muscle biopsies from diabetic patients.121 Similarly, myocardial and microvascular cells from streptozotocin-diabetic rats also show an upregulation of miR-503 expression with impaired angiogenesis, and the adenovirus delivery of anti-miR-503 to diabetic mice reverses these effects and improves postischemic angiogenesis.121

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MicroRNAs and Diabetes-linked Cardiac Disease

Typically, diabetic cardiac tissue phenotype displays hypertrophy, apoptosis, and cardiomyopathy. Reduced expression of miR-133 is seen in mouse and human models of cardiac hypertrophy and in cardiomyocytes exposed to HG are associated with poor contractility and cardiac hypertrophy.122,123 Data from another study suggest a possible link between miR-133 downregulation and prolonged QT interval in hearts from diabetic rabbits.124 Furthermore, a diabetic milieu is associated with increased levels of both miR-1 and miR-206 and correlated with downregulation of the anti-apoptosis factor, IGF-1, Pim-1, and heat shock protein 60 (Hsp-60), thus promoting cardiomyocyte apoptosis.125–127

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MicroRNAs and Diabetic Nephropathy

Diabetic nephropathy (DN) is an important diabetes-associated renal vascular complication, characterized by increased hypertrophic growth with accumulation of fibrotic extracellular matrix (ECM) proteins such as collagen and fibronectin in the renal mesangial cells (RMCs) along with kidney dysfunction.128 Based on data from both in vitro and in vivo models, a number of miRNAs have been shown to be upregulated (21, 26b, 29c, 192, 216a, 217, 200b/c, 215, 377) and downregulated (21, 25, 29a, 93, 192, 200a, 215, 451).129 Transforming growth factor β1 is a potent fibrotic inducer in DN, and miR-192 is upregulated in transforming growth factor β–treated mouse renal RMCs and in kidney cells from diabetic animals.130 However, in patients with established DN, miR-192 is downregulated.131 Independently, it has been shown that miR-192 regulates several downstream genes that include collagen type I alpha2 (Col1a2) and Col4a1, SMAD complex, and a series of downstream miRNAs (miR-216a/217 and miR-200b/c) that are reported to be major contributors to ECM accumulation, renal hypertrophy, and DN.132–134 miR377 is increased in human and mouse RMCs exposed to HG; results in up-regulation of fibronectin expression and downregulation of manganese superoxide dismutase and p21 activated kinase.135 miR-93 levels are lower in diabetic db/db mice kidney, podocytes, and renal microvascular ECs exposed to HG with parallel increases in VEGF-A expression.136 Decreases in miR-29c results in activation of Rho kinase by the targeting of Spry1 together with increases in the expression of collagen I, III, and IV, resulting in enhanced ECM accumulation and podocyte apoptosis.137,138

A number of miRNAs stand out as likely to be important in the pathogenesis of DN; however, their role may differ depending on the stage of DN. For instance, there is evidence that miR21 and miR192 are upregulated and downregulated with the downregulation of miR192 associated with patients with established DN.129

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MicroRNAs and Diabetic Retinopathy

Diabetic retinopathy is another microvascular complication of diabetes, characterized by alterations in the expression of vascular growth factors and angiogenesis in retinal ECs. miRNA expression profiling in retinal ECs from diabetic rats show that the NF-κB responsive miRNAs, miR-146, miR-155, miR-132, and miR-21, are upregulated in renal ECs in diabetes along with increased expression of VEGF and levels of p53-responsive miR-34. The overexpression of miR-146 has been shown to inhibit the NF-κB pathway, suggesting a negative feedback regulation by miR-146 on NF-κB activation in diabetic retinal ECs.139 miR-220b is downregulated in the retinas of diabetic rats and in HG exposed HUVECs and results in increased VEGF-A expression and angiogenesis. Furthermore, VEGF-A–mediated increases in vascular permeability and tube formation in HUVECs are reduced following transfection of the ECs with a miR-200b mimic.140 Finally, the upregulation of miR-29b141 and its potential target RAX, an activator of the pro-apoptotic PKR signaling pathway, has been implicated in apoptosis of retinal neurons and the development of diabetic retinopathy.

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In 2008, it was discovered that miRNAs are also present in blood, where they were detected in plasma, platelets, erythrocytes, and nucleated blood cells.142–144 We now know that hundreds of miRNAs are actively or passively released into the circulation and, subject to further validation, can be used to evaluate health status and disease progression.

Extracellular miRNAs circulate in the bloodstream and are remarkably stable. miRNAs can be packaged in microparticles (exosomes, microvesicles, and apoptotic bodies)145,146 associated with RNA-binding proteins [Argonaute2 (Ago2)]147 or lipoprotein complexes (high-density lipoprotein)148 to prevent their degradation. miRNAs may indeed function as mediators of cell-to-cell communication.145,149 Because of their stability in the circulation, miRNAs are currently being explored for their potential as biomarkers for CVD, for instance: myocardial infarction, heart failure, atherosclerotic disease, type 2 diabetes mellitus (T2DM), and hypertension. The precise cellular release mechanisms of miRNAs remain largely unknown; the earlier studies have revealed that these circulating miRNAs may be delivered to recipient cells, where they can regulate translation of target genes. However, we do not know if these circulating miRNAs have a specific active secretory pathway or they are passively released into circulation following cell damage or death. Circulating miRNAs may act in an endocrine-like fashion in the same way as peptide hormones.

The availability of biomarkers for the early detection of disease and the identification of individuals at risk of developing complications would greatly improve the health outcomes for these patients. Both T1DM and T2DM are associated with distinct modifications in the profile of miRNAs in the blood, which may be detectable several years before the disease manifests. Moreover, circulating levels of certain miRNAs may also be predictive of the risk of long-term complications. Thus, microvesicles in the blood that are derived from platelets, ECs, and monocyte/monocyte-derived cells and contain let-7 family, miR-17/92 family, miR-21, miR-29, miR-126, miR-133, miR-146, and miR-155 have been linked to metabolic and CVD.150 Furthermore, a number of miRNAs are deregulated before the onset of CVD and thus should be further investigated as candidate biomarkers for the early diagnosis of “pre-cardiovascular disease states,” such as obesity.151 Those circulating miRNAs that are associated with vascular diseases are summarized in Figure 4.

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An extensive database from studies using genetic approaches such as silencing and overexpression as well as pharmacological modulation of miRNAs indicates the importance of miRNAs in the pathogenesis of vascular disease and provides a unique opportunity to exploit the potential of miRNA-based therapeutics. miRNA therapeutics can be broadly classified based on the gain-of-function or loss-of-function of individual miRNA. At present, 2 methods are used to modulate miRNA activity: (1) miRNA mimics (restoring the function of a miRNA using either synthetic double-stranded miRNAs or viral vector-based overexpression and (2) miRNA inhibitors (inhibiting the function of a miRNA using chemically modified anti-miR oligonucleotides).152 Among the available methodologies, locked nucleic acid modified anti-miRNAs (called antogomiRs) have shown to be effective for the inhibition of miRNA. Likewise, adeno-associated virus vectors containing miRNA mimics have been found to be effective for the overexpression of the miRNA. However, the delivery of miRNA mimetics is more problematic than for miRNA inhibitors although several other methods including miRNA sponges (expressed as transcripts from plasmids/virus containing strong promoter and tandem repeats of binding sequences for a miRNA of interest), aptamers (conjugation of miRNAs or small hairpin RNA (shRNA) to engineered aptamer to be specifically delivered to a cell type), and polymer nanoparticles that can be engineered with miRNAs to recognize certain cell types are under investigations. Inhibitors can be classified either as small molecule inhibitors that are identified by cell-based screens or anti-miRs that inhibit miRNAs by complementary base pairing. The pharmacokinetic properties of antimiRs are still an area of uncertainty although their half-life is long and in the order of weeks.153 The results from several studies with miRNA inhibitors and/or overexpression using anti-miR oligos and mimics in various models of vascular disease indicate the potential benefits for the use of miRNAs (reviewed in detail128,153,154) as therapeutic agents to treat in CVD and diabetes are summarized in Figure 5. In the field of cancer therapeutics, the use of p53-regulated miRNAs that can upregulate the expression of the tumor suppressor, p53, is also a valuable approach that can be extended to other pathological states.155

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Metformin as a MicroRNA Modulator

Interestingly, several studies have provided evidence that metformin, the first choice oral hypoglycemic agent for the treatment of T2DM, modulates miRNA action not only in T2DM but also in cancer. Metformin treatment has been associated with the modulation of a series of miRNAs, including miR-26a, miR-192 and let-7c and miR-33a.156,157 Blandino et al156 have demonstrated that the RNase, DICER, is essential for the demonstration of the anticancer effects of metformin. Metformin induces the expression of DICER by a cellular mechanism mimicked by the AMPK activator, AICAR, and upregulates a number of miRNAs associated with energy metabolism, notably miR-33a. In contrast, Bao et al157 reported that in pancreatic cancer cells metformin downregulated a number of miRNAs: let-7a, let-7b, miR-26a, miR-101, miR-200b, and miR-200c. In the latter 2 studies, some of the data were generated with high concentrations of metformin (1–20 mM) that are not likely to be achieved with therapeutic doses of metformin. In contrast, it has been reported that pre-treatment of mouse microvascular ECs in culture with 50 μM metformin, which is within the upper limits of the therapeutic concentration range, prevents hyperglycemia-induced upregulation of miR-34a and downregulation of the decatylase SIRT1.158–160 The potential link between metformin, SIRT1, and the regulation of miRNA expression is of interest because treatment with metformin has been linked to reduced senescence and lifespan extension.161 miR-34a has been shown to induce EC senescence by the suppression of SIRT1.75,158–160 As previously discussed, miR-217 has also been described as an endogenous inhibitor of SIRT1 and lined to EC senescence83 emphasizing the importance of further investigating the links between miRNAs, the regulation of SIRT1 expression, and the treatment of diabetes-associated vascular disease.160,161

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The discovery of miRNAs as novel molecules that play important roles in the regulation of protein transcription/translation has opened up new avenues for biomedical research, particularly in the area of CVD. However, significant obstacles still exist for miRNA therapeutics to become a reality and, in particular, the challenges of specificity for a single cellular target, delivery of miRNA modulators, and pharmacokinetic considerations concerning cell permeability, absorption, distribution, and elimination. Nonetheless, the recognition of the important role that miRNAs play in disease processes is well recognized and undoubtedly we can look forward to significant advances in this field over the next few years.

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microRNA; cardiovascular disease; diabetes; endothelial cells; endothelial dysfunction; endothelial progenitor cell; vascular disease

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