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Abstract

Cardiovascular diseases are one of the most common causes of death in humans and are responsible for billions of dollars in health care expenditures. As the molecular basis of cardiac diseases continues to be explored, there remains the hope for identification of more effective therapeutics. MicroRNAs (miRNAs) are recognized as important regulators of numerous biological pathways and stress responses, including those found in cardiovascular diseases. MicroRNA signatures of cardiovascular diseases can provide targets for miRNA adjustment and offer the possibility of changing gene and protein expression to treat certain pathologies. These adjustments can be conferred using advances in oligonucleotide delivery methods, which can target single miRNAs, families of miRNAs, and certain tissue types. In this review, we will discuss the use of miRNAs in vivo and recent advances in their use for cardiovascular disease in mammalian models.

Keywords

cardiovascular disease microRNA therapeutics

Introduction

Cardiovascular disease (CVD) is the leading cause of mortality in the United States, and CVDs account for several of the leading causes of disability. This is in spite of numerous advances in pharmacologic and nonpharmacologic interventions.¹ Therefore, an improved understanding of the molecular mechanisms of CVDs and the methods to treat them are critical. Studies using microRNAs (miRNAs or miRs) to treat CVDs are becoming more popular and show promising results.²

MicroRNAs are short, noncoding nucleotides that are approximately 22 base pairs long and found in all metazoan eukaryotes.³ When recruited by the RNA-induced silencing complex, miRNA binds to the 3′-untranslated region (3′-UTR) of its target messenger RNA (mRNA) to silence translation and can occasionally tag the mRNA for degradation. The specificity of miRNAs for their targets is determined by a critical 5 base pair stretch termed the seed region, which is consistent among all members of each miRNA family. However, the nucleotides flanking the seed region on the miRNA do not need to be perfectly aligned with the surrounding mRNA sequence allowing multiple targets for any 1 given miRNA.³ Individual miRNAs have the ability to engage multiple mRNA targets, often encoding multiple elements of complex intracellular networks.⁴ Moreover, members of the same miRNA family often affect similar pathways.^5
-7 Thus, the manipulation of miRNA expression can have a profound impact on cellular phenotypes.

MicroRNAs have been shown to be important in many biological processes, including development and cancer.⁸ Not surprisingly, miRNAs have been found to play important regulatory roles in cardiovascular disease. Because of their highly conserved nature, it has even been theorized that miRNAs are important in evolutionary processes and biological network robustness.⁹ MicroRNAs are critical for healthy cardiovascular function and are involved in cardiogenesis and mediating proper cardiac conduction.¹⁰ As a demonstration of the overall importance of miRNAs in cardiac function, mice with a cardiac-specific knockout of Dicer, the endonuclease responsible for processing pre-miRNAs to their mature and functional miRNA form, developed dilated cardiomyopathy (DCM), heart failure (HF), and death. In this same report, the authors examined 4 patients with DCM and found decreased levels of Dicer compared with those in good cardiovascular health. Dicer expression increased after implantation of a ventricular assist device.¹¹

Because of their potential in vivo manipulability, miRNAs are now being investigated as therapeutic agents. Unlike most traditional therapeutic approaches in which drugs have specific cellular targets, the key component of miRNA modulation lies in the potential regulation of entire functional gene networks. However, since an individual miRNA may regulate numerous targets, the pathways that they modulate must be understood well before drugs can be fully developed, as off-target effects are a possibility.¹²

The involvement of miRNAs in several pathologies including hypertension and aortic aneurysm,¹³ atherosclerosis,¹⁴ and other diseases has been extensively reviewed,^15

-18 so we will focus on current progress in miRNA therapeutics for CVDs. This will include a brief discussion of some methods of miRNA transduction and delivery as well as pharmacokinetics, followed by recent studies that involve direct in vivo delivery or suppression of miRNAs in mammalian models of cardiovascular diseases.

MicroRNA Transduction and Delivery

Several techniques have been developed to deliver or inhibit miRNAs in specific target tissues or entire organisms without degradation, and this area has been reviewed previously (Figure 1).^2,18,19 The most common method of miRNA modulation is via “antagomirs,” which are oligonucleotides complementary to an endogenous mature miRNA and administered via injection.² Therapies can also be targeted to specific tissue types with adeno-associated viruses (AAVs).²⁰ Although numerous modifications have been developed in the delivery of miRNA modulators, there remain several important advantages and disadvantages among the common miRNA therapies (Table 1). Optimal dosing will likely depend on the tissue-specific content of each miRNA and the abundance of targets, as this may influence the effectiveness of both mimics and inhibitors.

Figure 1.

Methods of therapeutic microRNA delivery. Viral vectors can deliver RNA, which can code for mature microRNAs (miR). A mature microRNA binds to the RNA-induced silencing complex (RISC) to decrease the expression of a messenger RNA. MicroRNAs can be delivered as chemically modified mimics or antagomirs, which inhibit specific microRNAs. MicroRNA sponges can inhibit several microRNAs by binding them with complementary nucleotide sequences. LNA indicates locked nucleic acid; 2′-OMe, 2′-O-methyl; 2′-OMOE, 2′-O-methoxyethyl; 3′-UTR, 3′-untranslated region.

Table 1.

Advantages and Disadvantages of miRNA Therapeutic Delivery.

Therapy	Advantages	Disadvantages
Antagomir or mimic	Stable in vivo	Potential immune activation
	High efficiency	Potential liver accumulation
	High bioavailability	Dependent on delivery system
Viral vector	Tissue-specific delivery	Potential oncogene activation
Viral vector	Tissue-specific delivery	Potential immune response
Vesicle	High cargo versatility	Challenging purification and isolation
	Easy delivery to target tissues from vessels
	Potential immunological compatibility
Plasmid	Favorable immunogenicity	Difficult delivery to target tissue
miRNA sponge	Can effectively inhibit a family of miRNAs	Currently require viral delivery
miRNA sponge	Selectable marker or reporter gene vector	Limited in vivo studies

Abbreviation: miRNA, microRNA.

Antagomir Chemistries and Pharmacokinetics

Oligonucleotides like miRNAs must often be chemically modified to remain stable in vivo. Fortunately, many of these methods already existed before the discovery of miRNA and were used for applications that required stable oligoribonucleotides.²¹ Modified antagomirs are cleared from the plasma relatively rapidly after administration, compared with classical drugs.²² This is achieved by chemical modification such as conjugation to molecules like cholesterol, which greatly increases cellular uptake.

Antagomirs, or anti-miRNAs, may be complementary to a full sequence of a mature miRNA or just the seed region, both of which effectively downregulate the actions of an miRNA.^23,24 Stability of miRNAs can be achieved by chemical modifications like that of cholesterol conjugated via 2′-O-methyl (2′-OMe) or 2′-O-methoxyethyl (2′-OMOE) group linkages, with several phosphorothioate linkages.^15,24 More recently, locked nucleic acids (LNAs) have been developed, which improve stability by locking each ribose moiety with an extra bridge connecting the 2′-oxygen to the 4′-carbon.²⁵ The LNA antagomirs are often complementary to the mature 5′ region of an miRNA or just the seed region. However, the 2′-OMe and 2′-OMOE antagomirs are usually complementary to the whole mature miRNA sequence.¹⁵ Some studies have used mimics of mature miRNAs with similar chemical modifications to upregulate an miRNA,²⁶ but use of oligonucleotides for inhibition has been more extensively studied.

Newer antagomir chemistries are being developed like that of the miRNA “sponge.” This technique uses RNA with multiple miRNA binding site repeats that are complementary to the seed region of selected miRNAs. These binding site repeats can effectively “soak up” a given family of miRNAs decreasing their effectiveness via competitive inhibition. This inhibition could be made permanent via chromosomal insertion and delivery via viral vectors.²⁷

Viral Vectors for miRNA Delivery

Delivery of miRNAs to a certain cell type can also be achieved by viral vectors. Adeno-associated viruses can deliver miRNAs and have been used in murine models for CVDs like HF²⁸ and hypertrophy.²⁹ The availability of a variety of AAV serotypes allows for potential tissue specificity because of the natural tropism toward different organs of individual AAV serotypes as well as the different cellular receptors with which each AAV serotype can interact. The AAV serotype 9 (AAV9) has been shown to favor cardiac tissue in mice and rats and could therefore be effective for delivering miRNAs specifically to the heart.²⁰ Adeno-associated viruses are currently in use in a number of clinical trials for gene therapy, of which the safety profiles have not detected major adverse effects³⁰; however, there are no current trials involving miRNA and AAVs. Lentiviral vectors have also been used to both upregulate³¹ and downregulate³² miRNA expression but are much less frequently used because of concerns of integration into the host genome and possible oncogene activation.³³ Problems with viral vectors include possible poor transduction owing to biological barriers, but fewer adverse events are observed as additional serotypes, and mutants are developed.³⁴

Exosome, Apoptotic Body, and Microvesicle Transport

MicroRNAs can be transported via microvesicles³⁵ or exosomes³⁶ through the blood to exert their effects on different tissue beds. In addition, circulating miRNAs have been implicated in several CVDs.³⁷ The transport of miRNA via microvesicles or complexed to argonaute proteins, which may have the same properties as exosomes, is more common than miRNAs binding to high-density lipoprotein (HDL) or miRNAs circulating freely. Apoptotic bodies, which are microvesicles formed from cells undergoing apoptosis, have been used in therapy previously. Zernecke et al used apoptotic bodies derived from endothelial cells in a mouse model of atherosclerosis. They found that these apoptotic bodies had high levels of miR-126 and that they efficiently delivered the miRNA to the lesion sites, limiting the spread of atherosclerosis and stabilizing plaque size.³⁸ Further study found that after vascular endothelial injury, apoptotic bodies containing miR-126 from endothelial cells could promote vascular repair.³⁹ Another study used the same mouse model to administer microvesicles carrying miR-143/145 obtained from endothelial cells and found reduced atherosclerotic lesion formation.⁴⁰ These particles offer many advantages in their natural role in delivering biological information between cells, such as immunological compatibility. Therefore, this method of delivery holds promise for therapeutic use, with the potential to target vesicles to specific tissue beds.

Temporal Changes in miRNA Levels and Interactions Among miRNAs

MicroRNAs are part of complex biological pathways; therefore, any changes in miRNA levels, including temporal alterations, have the potential to have a large impact. However, the complex spatiotemporal pharmacodynamics of antagomirs and mimics may be difficult to study. Although miRNAs may be cleared from the blood quickly, they can remain in tissue for several weeks.⁴¹ This is problematic for the study of miRNA therapies because their effects on a pathway can sum over time and result in delayed effects. Montgomery et al found that although miRNAs tend to regulate their target genes quickly, an antagomir against miR-208a took several weeks to affect its target genes Myh7b and Myh7.⁴² Although tissues can be directly analyzed for miRNA levels in animal studies, possible studies with humans are difficult because the miRNAs are cleared from the serum so quickly. Therefore, accurate measures of the miRNA therapy in target tissue are difficult to quantify without tissue sampling. Further study on optimal miRNA therapy duration and timing is necessary.

In addition to miRNA sensitivity to duration of effect and dosage, miRNAs have been shown to cross talk with other miRNAs. Ling et al found that dickkopf 1 (DKK1) and phosphatase and tensin homolog on chromosome 10 (PTEN) were mutually regulatory via miRNA binding. Although gene silencing of 1 gene resulted in lowered expression of the other, inhibition of Dicer blocked this effect. Because the 3′-UTRs of both DKK1 and PTEN have several of the same miRNA binding sites, the miRNAs most likely compete with one another to regulate these 2 proteins.⁴³ These interactions further confound possible uses of miRNA therapeutics to bring about epigenetic changes.

MicroRNA Modulation Via Conventional Pharmacologic Therapy

MicroRNA modulation may also be achieved with more conventional pharmacologic therapies. For example, high salt-treated rats were found to have higher levels of miR-320 and lower levels of miR-26b and -21, according to microarray analysis of aortic tissue. These changes were ameliorated by administration of the β-blocker, nebivolol.⁴⁴ Further study found that nebivolol prevented a decrease in miR-27a/29a induced by a high salt diet in rats. Meanwhile, both atenolol, another β-blocker, and nebivolol prevented a decrease in miR-133a.⁴⁵ Statins, particularly atorvastatin, have also been found to prevent changes in miRNA expression in the diseased state.⁴⁶ Therefore, modulation of miRNAs from more conventional pharmacologic interventions, particularly oral medications that are more acceptable to patients, may also be an effective means of modulating miRNA levels and is worth further investigation.

Targeting miRNAs in Cardiac Diseases

Over the past several years, numerous animal models of cardiac disease have been developed and studied in order to identify the key mediators of cardiac pathology. Studies of changes in gene expression soon began to incorporate changes in miRNA expression. These same animal models now allow for the critical preclinical testing of miRNA modulators (Figure 2). Subsequently, we highlight relevant studies of individual miRNA therapeutics in a variety of disease states as summarized in Table 2.

Figure 2.

MicroRNA relationships with cardiovascular diseases. MicroRNAs have been used in therapeutic studies for several models of cardiovascular disease. Listed microRNAs have been used to modulate a molecular aspect of each indicated disease.

Table 2.

Cardiovascular Disease Model and miRNA Therapeutic Modulation.

Disease	MicroRNA	Modulation	Effect	References
Aneurysm	miR-21	Lentiviral overexpression	↑Cell proliferation, ↓apoptosis	⁴⁷
Aneurysm	miR-29b	Antagomir	↓Aneurism risk	⁴⁸
Angiogenesis	miR-24	Antagomir	↑Vascularization, ↓apoptosis	⁴⁹
Angiogenesis	miR-92a	Antagomir	↑Vascularization, ↓apoptosis	^50,51
Arrhythmia	miR-1	Antagomir	↓AF, ↑VT	⁵²
	miR-26	AAV overexpression, mimic	↓AF vulnerability	²⁶
	miR-328	Antagomir	↓AF maintenance	⁵³
Atherosclerosis/vascular remodeling	miR-21	Antagomir	↓Neointima formation	⁵⁴
	miR-33	Lentiviral antimiR-33, antagomir	↑HDL-C	^55,56
		Antagomir	↓ or no effect on plaque size	^{110, 111}
	miR-122	Antagomir	↓Cholesterol	^24,57,58
	miR-126	Apoptotic bodies	↓Lesion size, ↑vascular repair	^38,39
	miR-143/145	Microvesicles	↓Lesion size	⁴⁰
	miR-144	Mimic	↓HDL-C	⁵⁹
	miR-145	Lentiviral overexpression	↓Plaque size, ↑fibrosis	⁶⁰
		AAV overexpression	↓Neointima formation	⁸¹
Cardiogenesis	miR-1/133/208/499	Lentiviral overexpression	↑Fibroblast formation	⁶¹
Cardiogenesis	miR-302/367	Lentiviral overexpression	Fibroblast reprogramming	⁶²
Hypertrophy and fibrosis	miR-1	AAV9 vector	↓Hypertrophy, ↓fibrosis	²⁹
	miR-21	Antagomir	↓Atrial fibrosis	^63,64
	miR-25	Antagomir	↓Hypertrophy, ↓fibrosis	²⁸
	miR-27b	Antagomir	↓Hypertrophy	⁶⁵
	miR-29b	Antagomir	↑Fibrosis	⁷
	miR-34	Antagomir	↓Fibrosis	^66,67
	miR-101	AAV overexpression	↓Fibrosis	⁶⁸
	miR-132	Antagomir	↓Hypertrophy, ↓fibrosis	⁶⁹
	miR-133	AAV miR-133 precursor	↓Hypertrophy	⁷⁰
	miR-199b	Antagomir	↓Hypertrophy, ↓fibrosis	⁷¹
	miR-208a	Antagomir	↓Remodeling	⁴²
	miR-378	AAV9 vector	↑Cardiac function	⁷²
Infarct size	miR-15b	Antagomir	↓Infarct size	²³
	miR-17	Antagomir	↓Infarct size, ↑FS	⁷³
	miR-24	Lentiviral overexpression	↓Fibrosis, ↓Infarct size	⁷⁴
	miR-29a/29c	Antagomir	↓Infarct size, ↓apoptosis	⁷⁵
	miR-92a	Antagomir	↓Infarct size	⁵¹
	miR-99a	Lentiviral overexpression	↓Fibrosis, ↓apoptosis	⁷⁶
	miR-145	Antagomir	↑Infarct size, ↓FS	⁷⁷
	miR-210	Minicircle vector	↑FS, ↑neovascularization	⁷⁸
	miR-320	Antagomir	↓Infarct size	⁷⁹
Pulmonary hypertension	miR-17	Antagomir	↑Cell proliferation	⁸⁰
Pulmonary hypertension	miR-145	Antagomir	↑Cell proliferation	⁸¹

Abbreviations: AAV, adeno-associated virus; AAV9, AAV serotype 9; AF, atrial fibrillation; FS, fractional shortening; HDL-C, high-density lipoprotein cholesterol; miRNA/miR, microRNA; VT, ventricular tachycardia.

Cardiomyopathy and Hypertrophy

In response to pathological stimuli such as pressure or volume overload, the myocardium reacts with changes in gene expression resulting in cardiac remodeling. The sum effect of these adaptations leads to structural alterations in myocardial tissue and the extracellular matrix, which ultimately adversely effect cardiac performance.⁸² Cardiac hypertrophy is defined as an increase in ventricular mass caused by increased cardiomyocyte size and, in the context of cardiac disease, is an initial compensatory process that helps the heart to mitigate rising wall stress. However, this initial adaptive response becomes maladaptive as the chronic exposure to stress signals alters neuroendocrine activity and promotes the reexpression of fetal-type genes leading to impaired performance, characterizing HF.⁸³

There are several pathways that are now known to either promote or inhibit cardiac hypertrophy (eg, tumor growth factor β or nuclear factor κB), and although the exact molecular mechanisms that lead to it remain the focus of numerous studies, there are several miRNAs that are known to act within those pathways to affect hypertrophy.⁸⁴ Although the elucidation of those pathways would greatly help in determining the targets for miRNA therapies for hypertrophy, a large number of studies clearly indicate that miRNAs are crucial to those pathways and could be of therapeutic use.

Ganesan et al used AAV9 to exogenously express miR-378 in a chronic pressure overload model and a severe HF model via transgenic overexpression of β₁-adrenergic receptor. In the diseased state, miR-378 was downregulated. When subjected to transverse aortic constriction (TAC), mice that were treated prophylactically with AAV9-miR-378 exhibited improved cardiac function, a reduction in pulmonary congestion, and a decrease in mRNA levels of molecular markers of hypertrophy. The mechanism was thought to be via suppression of proteins in the MAP kinase signaling pathway.⁷² In another study, inhibition of miR-25 normalized TAC-induced cardiac dysfunction after establishment of LV dilation. The antagomir against miR-25 also improved fibrosis and cardiomyocyte cell size. Sarcoplasmic reticulum Ca²⁺ adenosine triphosphatase (SERCA2a), an established critical modulator of HF, was hypothesized to be the target of miR-25 because it was downregulated after introduction of miR-25 via AAV9 vector and was upregulated after anti-miR-25 administration.²⁸ Interestingly, current clinical trials with an AAV1 vector carrying SERCA2a in patients with advanced HF have shown promise without significant adverse effects.⁸⁵ Another TAC pressure overload model found that administration of an antagomir against miR-27b attenuated cardiac hypertrophy and dysfunction via upregulation of PPARγ.⁶⁵ Further, inhibition of miR-133 caused significant hypertrophy in healthy mice, while adenovirus-mediated introduction of an miR-133 precursor resulted in significantly smaller ventricular cardiomyocytes and no hypertrophy.⁷⁰

While the prospect of pretreating with an miRNA to reduce the risk of developing a pathology on the molecular level is intriguing, a better therapeutic approach would be to reverse a preexisting pathological condition to the healthy state. Karakikes et al recently showed that exogenous expression of miR-1 using the AAV9 vector in a rat model of pressure overload successfully reversed hypertrophy and reversed disease progression.²⁹ They observed a marked decrease in pathological remodeling as measured by cardiac fibrosis and mitogen-activated protein kinase (MAPK) signaling. Another study found that an antagomir against miR-199b reversed hypertrophy and fibrosis in HF mouse models. This was achieved via a normalization of Dyrk1a expression and reduced nuclear factor of activated T-cell (NFAT) activity, which are proteins implicated in HF.⁷¹ Similarly, miR-132 inhibition was found to suppress calcineurin and NFAT signaling after pressure overload and attenuate the development of hypertrophy.⁶⁹

Reprogramming

Since 2006, when Yamanaka and colleagues published on induced pluripotency,⁸⁶ mammalian cell fate plasticity has been intensely studied and garnered much more mainstream acceptance.⁸⁷ The cellular reprogramming field has seen explosive growth in both the number and scope of publications given the great potential for cardiac repair via reprogramming of fibroblasts into induced pluripotent stem (iPS) cells. Additionally, with the advent of iPS cell technology, patient-specific stem cells can provide a means to systematically study cardiogenic differentiation. Study of iPS cells from selected cohorts of patients is an innovative approach toward uncovering the molecular mechanisms of disease.

Several miRNAs have been shown to enhance iPS reprogramming when expressed along with combinations of the Oct4, Sox2, Klf4, and Myc transcription factors. The miRNAs miR-291-3p, miR-294, and miR-295 increased the efficiency of reprogramming by Oct4, Sox2, and Klf4.⁸⁸ In fact, it was found that the miR-302/367 cluster alone, administered via lentivirus, can reprogram fibroblasts more efficiently than the originally developed transcription factor method.⁶² This is important because one of the barriers to development of iPS therapeutics is efficiency, and the population of reprogrammed cells rarely reaches 10% with the best methods.

Repopulation of the injured heart with new, functional cardiomyocytes remains a daunting challenge for cardiac regenerative medicine with recent efforts directed toward conversion of injured areas into functional tissue. Reprogramming of mouse fibroblasts toward a myocardial cell fate by forced expression of cardiac transcription factors or miRNAs has recently been demonstrated. Nam et al demonstrated that 4 human cardiac transcription factors, including GATA binding protein 4, Hand2, T-box5, and myocardin, and 2 muscle-specific miRNAs, miR-1 and miR-133, activated cardiac marker expression in neonatal and adult human fibroblasts, reprogramming them into cardiac-like myocytes.⁸⁹

Jayawardena et al reported a method of reprogramming fibroblasts into cardiomyocyte-like cells with miR-1/133/208/499. Mice were subjected to ligation of left ascending coronary artery and injected intramyocardially with a lentiviral vector encoding the 4 miRNAs. They found that approximately 1% of cardiomyocytes in the infarcted hearts were reprogrammed fibroblasts, while a single cell was observed for the lentiviral negative control.⁶¹ Based on these reports, direct reprogramming and repair in vivo could be a possibility with the use of miRNAs. Nevertheless, there remain some concerns about both the use of iPS cells and reprogramming of fibroblasts due to their ability to form teratomas,⁹⁰ but the field is still developing.

Fibrosis

Numerous miRNAs have been studied in their relation to fibrosis, and these “fibromiRs” have recently been reviewed.¹⁶ FibromiRs have possibly the greatest therapeutic potential because numerous studies show reductions in fibrosis with few toxic side effects. In a myocardial infarction (MI) mouse model, lentiviral administration of miR-24 was found to reduce scar size after MI and reduce fibrosis extent.⁷⁴ A similar result was found with adenovirus-mediated overexpression of miR-101 in chronic MI rats.⁶⁸ In another MI mouse model, an antagomir against miR-21 was found to prevent atrial fibrosis.⁶³ This prevention of fibrosis via miR-21 antagomir was also observed by Thum et al and was attributed to inhibition of cardiac MAPK/extracellular signal-regulated kinase activity.⁶⁴ Moreover, a hypertension-induced HF rat model was mitigated by administration of an miR-208a antagomir. The miR-208a inhibition resulted in reduced cardiac remodeling, which significantly improved cardiac function.⁴² Furthermore, an antagomir against miR-15 was found to reduce infarct size and cardiac remodeling as well as enhance cardiac function in response to MI.²³ Finally, miR-34a was found by multiple groups to reduce fibrosis and increase angiogenesis in MI or pressure overload models.^66,67 Nevertheless, therapeutic targets need to be selected carefully or directed toward specific tissue beds because decreased fibrosis everywhere may lead to susceptibility to other ailments, like aortic aneurysm.

Angiogenesis

Angiogenesis is essential for cardiac repair following MI. Exciting preclinical studies evaluating proangiogenic therapies for MI have prompted the initiation of numerous clinical trials based on protein or gene transfer delivery of growth factors and administration of stem/progenitor cells, mainly from bone marrow origin.⁹¹ Not surprisingly, miRNAs have been found to play important roles in the modulation of angiogenesis and have been termed “angiomiRs.” One such angiomiR, miR-92a, is predicted to target several proteins involved in angiogenesis such as the integrin subunits α5 and αv, the histone deacetylase sirtuin 1, and Rap1. Overexpression of miR-92a in vivo prevented angiogenesis in a mouse model of both hind limb ischemia and MI. Meanwhile, an antagomir against miR-92a enhanced blood vessel growth possibly via upregulation of Itga5.⁵⁰ MicroRNA-92a has also been implicated in the recovery after MI in a large-animal model. Pigs were subjected to ischemia–reperfusion (I/R) injury and injected with an antagomir against miR-92a. The therapy resulted in significantly reduced infarct size, improved ejection fraction, and increased capillary density. The increased capillary density was also attributed to upregulation of Itga5.⁵¹ MicroRNA-24 was also found to be upregulated after cardiac ischemia. Injection of antagomirs against miR-24 in mice improved vascularization and cardiac function after MI via modulation of Gata2 and Pak4, which are involved in apoptotic and angiogenic processes.⁴⁹ Another method of miRNA therapy is via injection of minicircle vectors that contain miRNA precursors. Hu et al injected a minicircle vector carrying an miR-210 precursor intramyocardially after MI model induction. With this therapy, there was an increase in neovascularization, decreased apoptosis, and increased fractional shortening compared with control mice. The targets are hypothesized to be Efna3 and Ptp1b, which are involved in angiogenesis and apoptosis.⁷⁸

Myocardial Ischemia

Beyond angiogenesis, miRNAs have also been used to affect remodeling post-MI. Ren et al found that mice overexpressing miR-320 had larger infarct sizes and more apoptosis than those in wild-type controls. However, administration of an antagomir against miR-320 reduced infarction size relative to controls. The mechanism is mediated by upregulation of heat shock protein 20, a cardioprotective protein.⁷⁹ Another study found that lentiviral overexpression of miR-99a could attenuate injury (including fibrosis) after MI in a mouse model by preventing apoptosis and increasing autophagy via the mammalian target of rapamycin/P70/S6 K pathway.⁷⁶ In vivo inhibition of miR-17 via antagomir injection resulted in less matrix degradation and ventricular dilation as well as improved cardiac function possibly via restoration of myocardial tissue inhibitors of metalloproteinase (TIMP) 1 and TIMP2 after MI.⁷³ Recently, an antagomir against miR-145 was found to exacerbate infarct size, decrease fractional shortening, and decrease fractional area contraction. The mechanism of this effect is thought to be via a decrease in the differentiation of cardiac fibroblasts into myofibroblasts.⁷⁷ MicroRNA-29a/29c inhibition decreased apoptosis and infarct size and upregulated Mcl-1, an antiapoptotic Bcl-2 family member, which protected hearts from I/R injury.⁷⁵

Ischemic pre- or postconditioning involves exposure of the heart to repeated, short I/R episodes before or after a prolonged I/R episode, respectively. The role of miRNAs in preconditioning has been explored and may provide avenues for future therapy. Wang et al found that the miR-144/451 cluster was upregulated in mice that were preconditioned, and that an antagomir against miR-451 inhibited the cardioprotective effects of preconditioning.⁹² The mechanism is thought to be via inhibition of Rac-1-mediated oxidative stress signaling. In pre- or postconditioned rat hearts, significant miRNA expression changes were detected. After transfection of select miRNAs (miR-139-5p, 125b*, let-7b, and an antagomir against miR-487b) into cardiomyocytes during simulated I/R injury, there was significantly decreased cardiomyocyte cell death.⁹³ Another technique that has been found to confer cardioprotective effects is that of remote preconditioning. During remote preconditioning, episodes of I/R in tissues other than the myocardium render the myocardium more resistant to ischemia.⁹⁴ One study found that remote preconditioning with a blood pressure cuff on the upper arm in patients undergoing coronary artery bypass graft surgery had higher levels of miR-338-3p but lower levels of miR-1, compared with controls.⁹⁵ MicroRNAs can also be differentially expressed under pre-, post-, and remote preconditioning. Preconditioned rat hearts had higher levels of miR-1 and miR-21, while miR-1 was lower in post- and remote preconditioned hearts, compared with controls. Each conditioning group had significantly smaller infarct sizes, but the miRNA target proteins were not inversely correlated with the miRNA levels in each group.⁹⁶ Further study is necessary, but the role of miRNA in preconditioning is promising.

Atherosclerosis

MicroRNAs have been shown to play important regulatory roles in endothelial cells, vascular smooth muscle cells, and macrophage functions and thereby regulate the progression of atherosclerosis.¹⁴ Additionally, miRNAs may modulate several pathways implicated in plaque development such as cholesterol metabolism. Cholesterol is a key structural component of cell membranes, a precursor of many biologically important molecules including vitamin D, adrenal hormones, and sex hormones, and has been proposed to be involved in biological processes such as cell proliferation and neurological function. However, high cholesterol levels can cause accumulation within the walls of the endothelium triggering inflammation, creating flow limiting stenosis and decreasing elasticity by hardening the endothelial wall. Although statins have been shown to be effective in the treatment of coronary atherosclerosis and have significantly improved outcomes, there are still adverse events.⁹⁷ Therefore, the clinical manipulation of cholesterol metabolism pathways would be useful for the prevention of significant morbidity and mortality.¹ MicroRNAs are known to play many roles at varying stages in the metabolism of cholesterol from biosynthesis to dietary uptake and efflux.^98,99 Because so many miRNAs have been implicated in atherosclerosis, there is no dearth of suggestions for therapeutic targets^100

-103 or reviews on the subject of miRNAs and atherosclerosis.^{14,98,104,105}

One of the earliest described miRNAs was miR-122 owing to its high levels in the liver. Antagomirs against miR-122 were found to significantly lower cholesterol levels in both mice and nonhuman primates possibly by directly targeting the genes Ndrg3, AldoA, Bckdk, and Cd320.^24,57,58 In fact, miR-122 knockout mice have significantly lower cholesterol levels than those in control mice and this may be from lower microsomal transfer protein levels, which in turn lower very low-density lipoprotein secretion from the liver.^106,107

Another important miRNA linked with atherosclerosis is miR-33, which is implicated in modulation of adenosine triphosphate-binding cassette A1 (ABCA1) levels, among other proteins.^108,109 ABCA1 is a protein transporter involved in cholesterol efflux, so lower levels result in higher plasma HDL levels,⁹⁸ which has been observed both in mouse and nonhuman primate study with antagomirs.^55,56 In another study, the progression of atherosclerosis measured by plaque size and macrophage content was slowed by an antagomir directed against miR-33,¹¹⁰ although this was not corroborated by another study.¹¹¹ However, this may be due to the chemistry of the antagomir used as well as dietary differences between the mice.⁹⁸ ABCA1 is also targeted by miR-144, which was shown to lower HDL cholesterol levels when administered as an miRNA mimic.⁵⁹

Lovren et al found that miR-145 treatment via a lentiviral vector directed toward smooth muscle cells reduced plaque size in aortic sinuses, ascending aortas, and brachiocephalic arteries and increased fibrotic cap area.⁶⁰ In a rat balloon-injured carotid model, administration of an adenoviral miR-145 vector prevented the formation of neointima.⁸¹ A similar study found prevention of neointima formation after administration of an antagomir against miR-21 in the same rat model.⁵⁴ Therefore, there are several miRNAs that could be used in the prevention of atherosclerosis and neointima formation, although their profibrotic effects must be considered.

Arrhythmias

In the broad definition, an arrhythmia of the heart is any departure from normal sinus rhythm. Arrhythmias, especially ventricular arrhythmias, contribute greatly to CVD mortality. Therefore, the development of arrhythmias, particularly via calcium channel dysregulation from “rhythmiRs,” has been reviewed previously.^17,112

-115 However, the therapeutics of arrhythmogenesis using miRNAs remains in relative infancy compared with other CVDs.

One potential therapeutic target for treating arrhythmias is miR-328, which targets L-type Ca²⁺ channels. MicroRNA-328 was shown to be upregulated in both human rheumatic heart disease and a canine model of atrial fibrillation (AF). Adenoviral expression of miR-328 recapitulated the AF phenotype and an antagomir against miR-328 ameliorated the phenotype.⁵³

MicroRNA-1 has also been implicated in arrhythmogenesis and is one of the most commonly found miRNAs in cardiac tissue.¹¹⁶ Yang et al found that miR-1 is overexpressed in patients with coronary heart disease. In a rat model of MI, increased miR-1 levels resulted in ventricular tachyarrhythmias. This mechanism of arrhythmogenesis was possibly via repression of Kcnj2, which encodes the K⁺ channel subunit Kir2.1, and/or repression of Gja1, which encodes connexin 43. Administration of an antagomir against miR-1 into rats after MI ameliorated the ventricular arrhythmias.⁵² Another study found that patients with AF undergoing mitral valve repair or bypass grafting had significantly lower miR-1 levels and significantly higher Kir2.1 mRNA levels than those in control patients, possibly allowing for maintenance of AF.¹¹⁷ These findings are interesting because there appears to be a dosage of miR-1 required to maintain normal cardiac conduction, with significant elevations causing ventricular arrhythmias and reductions causing atrial arrhythmias. Further study revealed that miR-26 also targets Kir2.1 and is downregulated in animals and patients with AF. Vulnerability toward AF was ameliorated with administration of adenoviral miR-26 into mice. Meanwhile, antagomirs against miR-26 lead to increased Kir2.1 expression, and an injection of an miR-26 mimic into mice prevented AF associated with miR-26 knockout.²⁶ Therefore, both miR-1 and miR-26 are promising therapeutic targets for AF, and they appear to effectively target one of the same proteins.

Pulmonary Hypertension and Right Ventricular Failure

Pulmonary arterial hypertension (PAH) is a complex disease characterized by vascular remodeling with resultant elevation in pulmonary artery pressure and right ventricular failure. Although advancements in the pathogenesis and treatment of PAH have been made, it remains a severe and devastating disease with a poor prognosis and a high mortality, justifying the need of novel therapeutic targets. Dozens of miRNAs have been implicated in various forms of pulmonary hypertension.¹³ However, only a few have been tested for their therapeutic potential. In 1 mouse model of hypoxic PAH, miR-145 was found to be upregulated. Administration of an LNA antagomir against miR-145 showed significant protection against PAH development by increasing smooth muscle cell proliferation. In patients with PAH, miR-145 was found to be upregulated, indicating that antagomirs against miR-145 could be used to treat PAH.¹¹⁸ In another study with chronic hypoxia-induced PAH, miR-17, -21, and -92 were downregulated via antagomirs. Reducing miR-17 and -21 levels was found to reduce right ventricular systolic pressure but only miR-17 reduced the hypoxia-induced right ventricular hypertrophy. Additionally, monocrotaline-induced PAH rats saw significantly lower right ventricular systolic pressure and normalized cardiac output after administration of an antagomir against miR-17.⁸⁰

Aortic Aneurysm

The walls of the aorta may weaken from a variety of factors such as hypertension, inflammation, or a genetic susceptibility. If left untreated, the feared clinical consequence of aortic aneurysm is progression and acute rupture. In 2012, Maegedefessel et al demonstrated that treatment with an LNA inhibitor of miR-29b reduced the risk of aortic aneurysm in 2 murine models of abdominal aortic aneurysm (AAA). This was achieved by ultimately upregulating expression of collagen genes (Col1a1, Col3a1, Col5a1, and Eln) and generating an early fibrotic response in the abdominal aortic wall. Lentiviral overexpression increased AAA expansion.⁴⁸ Interestingly, another study examining patients with ascending thoracic aortic aneurysm found that several miRNAs were dysregulated including miR-29b upregulation¹¹⁹ and miR-21 downregulation.¹²⁰ In fact, miR-21 is one of the most commonly upregulated miRNAs in cancer and cardiovascular ailments¹²¹ and was found to ameliorate AAA in a murine model after lentiviral overexpression, while an LNA antagomir targeting miR-21 increased AAA size. The mechanism was via increased cell proliferation and attenuation of apoptosis, possibly through Pten. In human-explanted tissue, miR-21 levels were significantly higher in diseased aortic tissue than with those in control organ donor patients.⁴⁷ Therefore, treating AAA by altering miR-21 or miR-29b levels may be promising.

As highlighted by miR-29b, miR dysregulation can have disparate effects on organ systems and therefore, the downstream effects must be thoroughly studied. Lower miR-29b was found in aortic tissues of patients with AAA yet was increased in patients with dilated ascending aortas. Interestingly, inhibition of miR-29b in both cases prevented progression of disease. As mentioned earlier, fibrosis is often considered to be a pathologic consequence and can be accompanied by a significant impact on the affected organ system. Systemic downregulation of miR-29b, with potential fibrosis in other organs, would not be useful for translational approaches in humans; however, catheter-based local delivery of anti-miR-29b may emerge as a promising avenue to trigger fibrosis only in the aortic wall in humans. A biological process with seemingly disparate effects, such as fibrosis, highlights the importance of precise delivery of miRNAs, optimal dosing, and an understanding of potential off-target effects.

Conclusion

The tools to study miRNAs are becoming more sophisticated and readily achievable¹²²; however, questions remain about the effectiveness of antagomirs and viability of viral reintroduction of miRNAs. Currently, there is 1 anti-miR chemistry in clinical trials, which is miravirsen (an inhibitor of miR-122). Phase I and II clinical trials of miravirsen for the treatment of hepatitis C virus seem promising.¹²³ However, no clinical trials have begun using miRNA therapy for CVDs. There remain numerous questions in the development of miRNA-based therapeutics for any CVD process. To date, animal studies of systemic therapies remain focused in the target tissue of interest, without thorough evaluation of effects in other organ systems. Beyond this critical step, the appropriate dosing regimens and duration of effect need to be established. Beyond defining the correct dosing regimen, finding the correct timing of delivery for the specific pathologic process will also need to be studied. As outlined in this review, nearly all aspects of CVD are subject to miRNA manipulation; however, this novel therapy comes with numerous advantages and disadvantages (Table 3).

Table 3.

Advantages and Drawbacks of miRNA-Based Therapeutics.

Advantages	Drawbacks
Potent effects from relatively small doses	Delivery, particularly to specific tissues, is difficult
Effects pathways rather than specific targets	Effects of miRNAs can be therapeutic or harmful depending on the tissue (eg, fibrosis)
Specific miRNAs are easily regulated	Off-target effects are likely
Sustained effects	Possible toxicity of delivery vectors or miRNAs themselves
	Tissue-specific pharmacodynamics difficult to determine

Abbreviation: miRNA, microRNA.

The treatment of advanced HF has garnered significant attention as the number of individuals affected and the health care expenditures used to treat them continue to rise. Given the multiple molecular targets that are now known to effect cardiac remodeling, it is not surprising that novel therapies for HF are being brought into clinical trials. Novel therapies for HF have shown promise and appear poised for advancement in clinical trials as evidenced by current clinical gene therapy trials.¹²⁴ Based on the numerous animal studies, several candidate miRNAs, such as miR-25, may also move quickly into clinical trials.

In miRNA therapeutics, off-target effects are always problematic as each miRNA has numerous targets. Therefore, the targeting of therapy to specific tissues (eg, with AAVs) should be further investigated. As technology improves both in the realm of molecular targeting and percutaneous delivery methods, issues of systemic delivery can be mitigated. Regardless, careful study is necessary as off-target effects even within the target organ may not be obvious. Overall, the field of miRNA therapeutics is promising, particularly in cardiovascular health, and there is no shortage of possible miRNA targets for study.

Footnotes

Author Contributions

Calway, T contributed to conception and design, contributed to analysis and interpretation, drafted the manuscript, critically revised the manuscript, gave final approval, and agrees to be accountable for all aspects of work ensuring integrity and accuracy. Kim, G contributed to conception and design, contributed to analysis and interpretation, drafted the manuscript, critically revised the manuscript, gave final approval, and agrees to be accountable for all aspects of work ensuring integrity and accuracy.

Declaration of Conflicting Interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: Work is supported by NIH-K08-HL098565 (GHK) and the Institute for Cardiovascular Research, University of Chicago.

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Harnessing the Therapeutic Potential of MicroRNAs for Cardiovascular Disease

Abstract

Keywords

Introduction

MicroRNA Transduction and Delivery

Antagomir Chemistries and Pharmacokinetics

Viral Vectors for miRNA Delivery

Exosome, Apoptotic Body, and Microvesicle Transport

Temporal Changes in miRNA Levels and Interactions Among miRNAs

MicroRNA Modulation Via Conventional Pharmacologic Therapy

Targeting miRNAs in Cardiac Diseases

Cardiomyopathy and Hypertrophy

Reprogramming

Fibrosis

Angiogenesis

Myocardial Ischemia

Atherosclerosis

Arrhythmias

Pulmonary Hypertension and Right Ventricular Failure

Aortic Aneurysm

Conclusion

Footnotes

Author Contributions

Declaration of Conflicting Interests

Funding

References