A MicroRNA Perspective on Cardiovascular Development and Diseases: An Update

In this review, we summarize the latest research pertaining to MicroRNAs (miRs) related to cardiovascular diseases. In today’s molecular age, the key clinical aspects of diagnosing and treating these type of diseases are crucial, and miRs play an important role. Therefore, we have made a thorough analysis discussing the most important candidate protagonists of many pathways relating to such conditions as atherosclerosis, heart failure, myocardial infarction, and congenital heart disorders. We approach miRs initially from the fundamental molecular aspects and look at their role in developmental pathways, as well as regulatory mechanisms dysregulated under specific cardiovascular conditions. By doing so, we can better understand their functional roles. Next, we look at therapeutic aspects, including delivery and inhibition techniques. We conclude that a personal approach for treatment is paramount, and so understanding miRs is strategic for cardiovascular health.


MicroRNA's
Initial studies by Ruvkon et al. and Ambros et al. in the 1990s demonstrated that a group of small non-coding RNAs influenced the development of Caenorhabditis elegans by regulating translation thorough a process of base-pairing (inhibiting translation) to the 3 UTR and, in a few cases, the 5 UTR of mRNA [1,2]. These small RNAs, termed microRNAs, were over time shown to be conserved in many cellular eukaryotic species including human cells [2][3][4][5].
MicroRNAs (miRs), commonly designated miR for the precursor product and miR for the mature product, are a set of 18-24 nucleotides which, when processed (a process of complementary base pairing to mRNA), can silence or downregulate expression of their targets. Their function is to either destabilize and/or cleave the target mRNA, or reduce the efficiency with which the mRNA can be processed [6,7]. Unlike other small RNAs, miRs have a predictable hairpin loop structure from their precursor transcript [8]. This small region, required for miRNA regulation, is only about eight nucleotides long. Such a small region, potentially homologous in many places, suggests that one miR regulates as much as 60% of available mRNAs, although this has been found not to be the case [9]. MiRNA translation regulation is a tightly regulated mechanism that is controlled in a spatial-temporal manner, meaning that a handful of miRs can be secreted in specific organs and are taken up far from their origin [5,6,10].

miRNA Biogenesis
Briefly, miRs are initially transcribed from the genomic region by pol II (up to 3 kb) as a combined primary-precursor miR (pri-miR). Further processing by Drosha/DGCR8 permits cleavage Figure 1. mRNA biogenesis. Transcription of a pri-miR by pol II, followed by cleavage (Drosha/DGCR8 complex) to a pre-miR. The pre-miR is later exported to the cytoplasm via exportin-5/Ran-GTP, where it can be further cleaved by a Dicer/TRBP complex and unwind into its mature form. This is further packed by AGO into the RNA-induced Silencing Complex (RiSC) with mRNA. The consequence of such loading is either a transcriptional suppression or transcript degradation.
Given the above, it is no surprise that miRs are such important regulatory molecules. In many instances, they self-regulate by co-expression with target genes. As a result, many research groups have dedicated a considerable amount of time and resources to studying and profiling miRs and their role in development, as well as diseases caused by miR mis-expression, errors in transcription, promoter defects (such as hypo-or hyper-methylation), and others [5,8,[15][16][17][18]. Note that the remainder of this review refers to the functional properties of miRs.

miRNAs in Cardiomyocytes
Fundamentally, the adult heart has little potential to regenerate when suffering injury or disease. Normally, cardiomyocytes are lost, leading to heart failure and, in more complicated cases, death [19][20][21]. Hence, understanding cardiomyocyte development has been key to understanding repair.
Studies of individual miRs using developmental models of the heart have led to the discovery that miR-1/miR-133 are fundamental to both the control of proliferation as well as the regulation of muscle transcriptional networks, e.g., SRF, MEF2c, MyoD, Hand2, and Myocardin. Interestingly, SRF has been found to be a requirement for miR-1 expression during development. This double mechanism is particularly evident, as SRF/miR-1 also come together to regulate the sodium-calcium exchanger NCX1 [22]. Additionally, miR-1 overexpression blocks ventricle myocyte expansion [23]. Furthermore, miR-1 has been linked to the NOTCH1 receptor. Its ligand, Dlk-1, is a critical factor in specification through asymmetric division [24]. Mouse studies by Wu et al. found evidence that miR-34a is a repressive regulator to NOTCH1, while upregulating Jagged1, Hey2, and Hes [25]. A variant of miR-1-1 in mice is miR-1-2, and its deletion has been found to be lethal to approximately 50% of Figure 1. mRNA biogenesis. Transcription of a pri-miR by pol II, followed by cleavage (Drosha/DGCR8 complex) to a pre-miR. The pre-miR is later exported to the cytoplasm via exportin-5/Ran-GTP, where it can be further cleaved by a Dicer/TRBP complex and unwind into its mature form. This is further packed by AGO into the RNA-induced Silencing Complex (RiSC) with mRNA. The consequence of such loading is either a transcriptional suppression or transcript degradation.
Given the above, it is no surprise that miRs are such important regulatory molecules. In many instances, they self-regulate by co-expression with target genes. As a result, many research groups have dedicated a considerable amount of time and resources to studying and profiling miRs and their role in development, as well as diseases caused by miR mis-expression, errors in transcription, promoter defects (such as hypo-or hyper-methylation), and others [5,8,[15][16][17][18]. Note that the remainder of this review refers to the functional properties of miRs.

miRNAs in Cardiomyocytes
Fundamentally, the adult heart has little potential to regenerate when suffering injury or disease. Normally, cardiomyocytes are lost, leading to heart failure and, in more complicated cases, death [19][20][21]. Hence, understanding cardiomyocyte development has been key to understanding repair.
Studies of individual miRs using developmental models of the heart have led to the discovery that miR-1/miR-133 are fundamental to both the control of proliferation as well as the regulation of muscle transcriptional networks, e.g., SRF, MEF2c, MyoD, Hand2, and Myocardin. Interestingly, SRF has been found to be a requirement for miR-1 expression during development. This double mechanism is particularly evident, as SRF/miR-1 also come together to regulate the sodium-calcium exchanger NCX1 [22]. Additionally, miR-1 overexpression blocks ventricle myocyte expansion [23]. Furthermore, miR-1 has been linked to the NOTCH1 receptor. Its ligand, Dlk-1, is a critical factor in specification through asymmetric division [24]. Mouse studies by Wu et al. found evidence that miR-34a is a repressive regulator to NOTCH1, while upregulating Jagged1, Hey2, and Hes [25]. A variant of miR-1-1 in mice is miR-1-2, and its deletion has been found to be lethal to approximately 50% of embryos, while around 20% of survivors have major cardiac defects. Moreover, its deletion has also been reported to repress Kcnd2, a crucial factor in repolarization of the heart [26][27][28]. In adult rat experiments, miR-1 has been also revealed to target KCNQ1 and KCNE1 [18].
Meanwhile, the miR-17-92 cluster has been implicated in cardiac proliferation by negatively regulating the PTEN tumor suppressor gene [9,29,30]. Additional reports suggest that miR-17-92 cluster overexpression could cause Tsc1 repression. Consequently, it causes mTOR-mediated hypertrophy, inferring that the conditional downregulation of Tsc1 acts as a negative regulator of mTOR and a downstream target of PTEN [31]. Furthermore, the miR-15 family seems to balance proliferation by repressing cell cycle regulators, in particular through decreasing HSP-20, targeting Bcl2, and repressing TGFβ activity [30].
Finally, a distinguishing trait of cardiomyocytes is their contraction. This is provided in part by a delicate balance of α/β-MHC, chiefly controlled by miR-208. Initially, miR-208a was found to regulate GATA4 and CX40, thereby partially regulating the conduction system. In addition, Callis et al. found that both isoforms of miR-208 (a/b) target TRAP1 and myostatin, which are important negative regulators of muscle growth and hypertrophy [32,33] (a synopsis of knowledge about miR regulation during heart development is shown in Table 1).  [33,37,39,40] miRNAs in Stem Cells to Cardiomyocyte Differentiation In order to bypass limitations for cardiomyocyte regeneration, techniques such as direct differentiation of somatic cells, and use of induced pluripotent stem (IPS) cells and embryonic stem (ES) cells, have been the focus for many groups for the past two decades, and still continue to be a large field of study within organ repair [33,41].
Direct differentiation of fibroblasts to cardiomyocytes was initially achieved using the transcription factors Hand2, GATA4, MEF2c, and TBX5 [42], and later enhanced by the addition of MYOCD, SRF, SMARCD3, and MESP1, yet they never addressed the role of miRs [33,39,43]. Nonetheless, by pioneering the use of miRs, Dzau´s group began to show the potential of miR-1, by demonstrating that its overexpression was sufficient to convert cells [40].
Expression of miR-1/-499 in ES cell-derived cardiomyocytes predominantly controls the electrical/conduction system. Their expression upregulates Kir2.1, Kv1.4, HERG, and DHPR while downregulating HCN4, resulting in cardiac potassium current channel increase I to , I ks , and I kr , and decreased I f [18,38], a result consistent with the expression of the RYR2 L-type channel [44]. In particular, miR-499 has been implicated in the expression of MHC6, MHC7, MLC2, and TNNT2 [33].
Poon et al. recently described the importance of miR-200c using ES cells, denoting GATA4, TBX5, and SRF to be its targets [36]. They noted that knockouts in ES cells altered Ca + , Na + , and K + ion channels (CACNA1C, KCNJ2, and SCN5A), increasing contractility [45]. Additionally, conduction seems to be altered if miR-1(-2) is deleted, via dysregulating the expression of Irx 4, 5, and Kcnd2. Meanwhile, transcription factor MESP1, once described as the key regulator of the heart by Bondue and Blanpain (2010), regulates Nkx2.5, Tbx5, Hand2, FoxH1, Isl1, and others, while at the same time inducing mesoderm lineage by blocking Bry, Fgf6, FoxA2, Sox17, and Gsc [46]. Thereby, MESP1 sits atop of the hierarchy of cardiomyocyte formation and regulation [47]. At an earlier stage, we find that transactivation is effected by miR-322/503 by targeting the RNA binding factor Celf1, which would otherwise induce a neural fate [37]. Regulation via the most important miRs during differentiation can be seen in Figure 2.
Poon et al. recently described the importance of miR-200c using ES cells, denoting GATA4, TBX5, and SRF to be its targets [36]. They noted that knockouts in ES cells altered Ca + , Na + , and K + ion channels (CACNA1C, KCNJ2, and SCN5A), increasing contractility [45]. Additionally, conduction seems to be altered if miR-1(-2) is deleted, via dysregulating the expression of Irx 4, 5, and Kcnd2. Meanwhile, transcription factor MESP1, once described as the key regulator of the heart by Bondue and Blanpain (2010), regulates Nkx2.5, Tbx5, Hand2, FoxH1, Isl1, and others, while at the same time inducing mesoderm lineage by blocking Bry, Fgf6, FoxA2, Sox17, and Gsc [46]. Thereby, MESP1 sits atop of the hierarchy of cardiomyocyte formation and regulation [47]. At an earlier stage, we find that transactivation is effected by miR-322/503 by targeting the RNA binding factor Celf1, which would otherwise induce a neural fate [37]. Regulation via the most important miRs during differentiation can be seen in Figure 2. Comprehending the role of miRs in all aspects of the cardiovascular system is in itself a formidable task that warrants further in-depth discussion in a variety of areas. The goal of this review is to inform the reader about a handful of clinically relevant diseases in which miRs not only play a crucial role but in which their directive could be key in regulating or attenuating them. We will also briefly allude to other aspects of cardiac miR regulation. Concisely, miR-21's function during stress is one of the most studied. It is known that miR-21 up-regulation represses Spry1 [48,49], leading to PI3K/Akt signaling and increased Matrix metallopeptidase 2 (MMP-2) expression via PTEN repression. As a corollary, TGF-β1 seems to be regulated by Spry1, also as a response to Ang II in atrial fibroblasts [49,50]. Fibrosis, an outcome of these signaling cascades, seems to also be Comprehending the role of miRs in all aspects of the cardiovascular system is in itself a formidable task that warrants further in-depth discussion in a variety of areas. The goal of this review is to inform the reader about a handful of clinically relevant diseases in which miRs not only play a crucial role but in which their directive could be key in regulating or attenuating them. We will also briefly allude to other aspects of cardiac miR regulation. Concisely, miR-21's function during stress is one of the most studied. It is known that miR-21 up-regulation represses Spry1 [48,49], leading to PI3K/Akt signaling and increased Matrix metallopeptidase 2 (MMP-2) expression via PTEN repression. As a corollary, TGF-β1 seems to be regulated by Spry1, also as a response to Ang II in atrial fibroblasts [49,50]. Fibrosis, an outcome of these signaling cascades, seems to also be exacerbated by the fact that miR-21, encapsulated in exosomes, is excreted by the proliferating fibroblasts [48,51]. Moreover, the family of miR-29 seems to be the opposite. During and after cardiovascular disease stress, the miR-29 family peaks in its function, promoting expression of collagens, fibrilins, and elastins-all factors involved in fibrotic proliferation. Its knockdown has been independently confirmed to upregulate collagens in the heart [5,9]. Another interesting member is miR-133, which maintains a balance in the expression of connective tissue. Thus, downregulation of this factor stimulates the extracellular matrix [9]. In addition, it seems to be co-expressed with miR-30. Studies have shown that during hypertrophy or heart failure; the expression of both factors is decreased alongside connective tissue growth factor [48,52].
Inflammatory response in the heart is characterized by a myriad of events in which monocytes, macrophages, and leukocytes participate. As a result, there is a production of pro-and anti-inflammatory cytokines such as Tumor necrotic factor α (TNF-α), and Interleukin 1, 6 (IL-1, -6) [53,54]. MiRs are involved in the signaling cascades, of which some of the best studied include miR-155 and miR-233, which inhibits NF-κβ via repression of IKK-β. Systemic administration has been proven to mitigate dysfunction and improve survival by targeting JNK. Nevertheless, miR-155 knockdown triggers apoptosis via induced liposaccharides [45,55,56]. In concert with miR-155, miR-146 downregulates expression of IRAK1 and TRAF6, which are upstream activators of NF-κβ [5]. Additionally, IL-6 and collagen expression appears to be regulated by let-7i-5p. Let-7i-5p activation reduces general inflammation by downregulating IL-6. IL-6, along with NLRP3, is mediated by miR-233 in monocytes [45]. Further, during myocarditis, miR-155 upregulates macrophages and CD4 + T lymphocytes [55]. Another strategic player during inflammation is miR-126, due to its control over endothelial VCAM-1, which in turn help controls leukocyte trafficking and vasculature inflammation, potentially leading to cardiac repair by affecting the homing of hematopoietic progenitor cells [9,45].

miRNAs in Cardiovascular Diseases
Cardiovascular Diseases (CVDs) are the leading cause of morbidity and mortality in developed countries. Over the past two decades, much effort has been put into finding both the physiological fundamentals and molecular mechanisms of control for CVDs. Research on miRs in CVDs has pointed out the specificity of certain miRs and clusters to certain conditions. Here, we emphasize on some of the most important and clinically relevant miRs in conditions such as congenital heart disease (CHD), atherosclerosis, myocardial infarction (MI), severe coronary artery disease (CAD), and heart failure (HF) [4,9,15,33,57] (see Figure 3 and Supplementary Tables S1 and S2).
CHDs are the leading cause of prenatal deaths (~40%), while at the same time comprising the majority of all congenital malformations [58]. Reports from the Euro Heart Survey suggest that around 20% of patients with CHD undergo surgery or a catheter-based intervention, resulting in a major economic burden for the patient [15,33,59,60].
Ventricular septal defect (VSD) accounts for approximately 30-40% of CHDs, whereas atrial septal defects (ASD) are the cause of a smaller proportion. VSD and ASD can be viewed as a discontinuation in the septal wall dividing the ventricles and atria, respectively, of the heart. VSD can produce left ventricle overload, resulting in pulmonary hypertension [61,62]. VSD elevates levels of GJA1 and SOX9 overlap with reduced expression of miR-1-1, and elevates miR-181c [15]. MiR-1/181c additionally regulate the expression of Bone morphogenic receptor protein 2 (BMPR2), a key component in energy biogenesis [63,64]. Additional data form Li et al. showed that let-7e-5p, miR-222-3p, and miR-433 maybe the underlying cause for abnormalities since they target NOTCH1, HAND1, GATA3, and ZFPM2, resulting in altered morphogenesis and VSD [27].

miRNAs in Cardiovascular Diseases
Cardiovascular Diseases (CVDs) are the leading cause of morbidity and mortality in developed countries. Over the past two decades, much effort has been put into finding both the physiological fundamentals and molecular mechanisms of control for CVDs. Research on miRs in CVDs has pointed out the specificity of certain miRs and clusters to certain conditions. Here, we emphasize on some of the most important and clinically relevant miRs in conditions such as congenital heart disease (CHD), atherosclerosis, myocardial infarction (MI), severe coronary artery disease (CAD), and heart failure (HF) [4,9,15,33,57] (see Figure 3 and Supplementary Tables S1 and S2). CHDs are the leading cause of prenatal deaths (~40%), while at the same time comprising the majority of all congenital malformations [58]. Reports from the Euro Heart Survey suggest that around 20% of patients with CHD undergo surgery or a catheter-based intervention, resulting in a major economic burden for the patient [15,33,59,60]. DiGeorge syndrome is a direct result of deletion in region 8 of chromosome 22, leading to a loss-of-function mutation on TBX1 and haploinsufficiency.
TBX1 is implicated in neural crest cell differentiation, in which mutations to the gene hinder correct formation of the outflow track [15]. In addition, this condition leads to DGCR8 downregulation and promotes an accumulation of both pri-miRs and pre-miRs [65].
Recent studies have shown 5 miRs to be directly correlated with Down syndrome (DS; CHD expectancy~50-60%): miR-99a, let-7c, miR-125b-2, miR-155, and miR-802, all overexpressed in the heart [51]. MiR-99a has been associated with repression of cardiogenesis when expressed at an early stage, by regulating Smarca5. Let-7c was found to induce cardiogenesis, but only if expressed during mesoderm formation, thereby repressing the activity of Ezh2 [66]. Additional studies in cancer have concluded that the loss of the let-7 family contributes to the upregulation of Ezh2, while miR-99a, typically found in patients with prostate cancer, can contribute to general cell proliferation [66]. Moreover, miR-155 overexpression can inhibit necrosis. Additional survival studies that suggest a mechanism via repression of receptor interacting protein 1 (RIP1) are independent of both the Wnt/β-catenin and Akt survival pathways [15,56,67]. MiR-155 is involved in many known aspects of regulatory biology: promoting cell proliferation by PTEN signaling pathway [29], promoting tumor growth by ARID2 repression [68], and regulating cell proliferation in glioma by targeting FOXO3a [69].
According to the AHA website (http://www.heart.org/, accessed on 20 April 2018) "Atherosclerosis is a big word for a big problem". They define atherosclerosis as a buildup of fat deposits turning into plaques in the arteries. It is a multidimensional problem, not only dependent on the amount of circulating fat, but also on factors such as endothelial cell (EC) dysfunction, and vascular smooth muscle cell (VSMC) differentiation and inflammation. These buildups can lead to partial or full blockage and thus restrict blood flow, nutrition, and/or oxygen. Consequently, it is the initiator for many diseases, including CHD, angina, and carotid artery disease, amongst others [70]. A major component of atherosclerosis is EC dysfunction as a response to sheer stress. Schober et al. deduced that miR-126 directly affects vascular integrity, leading to the notion that miR-126-5p is mainly responsible for EC repair by inhibiting NOTCH1 and Dlk1, [71]. Additionally, a second isoform, miR-126-3p, is responsible for reducing inflammation signaling by promoting VCAM-1.
By blocking these two mechanisms, atherosclerosis protection at the EC level is reduced [72,73]. Romano's group uncovered the mechanics of understating the cascade activation of miR-126-5p by Lipoxin A4-a response via pro-inflammatory endothelial microvesicles packed with miR-126-5p, which antagonizes TNFα, leading to the upregulation of VCAM-1 and the downregulation of SPRED1 [71]. Another component of atherosclerosis is VSMC differentiation. MiR-145 deficiency has been shown to reduce the medial layer in arteries. In addition, the differentiation genes myocardin, KLF4, KLF5, calmodulin kinase, and cholesterol transporter ABCA1 were found to be a direct target of miR-145 [74].
MiR-33a and miR-33b have been shown to regulate ABCA1 and ABCG1, since they control the sterol regulatory element-binding proteins. Hence, their control can be a useful tool in potential therapies for dyslipidemia and atherosclerosis [75,76]. Hypertension or high blood pressure can lead to atherosclerosis; due to the added force at the artery walls, miR-145 and miR-143 seem to play an important role in high blood pressure, mainly targeting the angiotensin converting enzyme [4]. In addition, inhibition of miR-145 might improve diabetic resistance via nitric oxide [77]. We note that mouse studies have elucidated the role of miR-21, demonstrating reduced blood pressure in inverse correlation with miR-130a and miR-195, which have been shown to be positively upregulated in serum [78].
MI can be described as severe CAD or a myocardial cell death due to sustained ischemia. Patients with MI have shown higher levels of expression of miR-1, miR-133, miR-208, and miR-499 [79]. The dysregulation of all four miRs has been linked to MI [18,33,79,80]. MiR-208 by itself has been shown to be sufficient to induce heart hypertrophy as a response to overload while inducing β-MHC expression [81]. As mentioned in Sun et al., levels of expression of miR-1, miR-16, miR-21, miR-92a, miR-195, miR-208, miR-375, miR-494, miR-103, miR-107, miR-325, and miR-874 are appreciably upregulated in heart tissue MI, while the levels of tissue miR-133a/b, miR-214, miR-873, miR-2861, miR-30b, miR-188-3p, and miR-145 are decreased. These miRs contribute to the notion of specific spatial-time regulation, since many (if not all) are also involved in other conditions. In addition, protective signaling to reduce damage in the heart can be achieved by expression of miR-873 and miR-2861 [82]. The specificity required to precisely achieve protection in the midst of so much disruption can tell us more about the recurrent self-protective and pro-survival mechanisms present in the heart. It was mentioned that MI constitutes severe cell death, and cell death itself comes in 3 "flavors": necrosis (necroptosis), autophagy, and apoptosis [82][83][84]. Each comes with a set of miR regulators acting on specific targets to both promote and inhibit each process.
Necrosis is a known form of death due to exacerbation in cellular or pathogenic damage. In cardiomyocytes, necrotic death induced by O 2 elevates levels of miR-103 and miR-107, which act on the Fas-associated protein with death domain [85]. Meanwhile miR-874 expression can lead to necrosis by activating FOXO3a and Caspase-8 [86]. Recently, research has revealed a form of necrosis via programmed death: necroptosis. This process is initiated by TNF-α with directed interactions to RIP (1 and 3) proteins. Briefly, deficiency in interferon-β and MLK, as a result of TNFα/RIP, induce pyruvate dehydrogenase to induce what is now referred to as the necrosome (independent of caspase-8). However, this pathway can be mediated by activation of miR-874 [54,87]. Additionally, miR-155 can block RIP1 interaction, inhibiting the necrosome [29,56].
Autophagy is a highly conserved process of delivering intracellular components, including mitochondria and long-lived macromolecules, via a double-membrane structure, to the lysosomes for degradation [83]. When activated, miR-188-3p, miR-290, and miR-375 act as mediators reducing autophagitic activity by activating ATG7. ATG7 acts through a thiol-ester bond on the E1 activator to free ubiquitin molecules, beginning degradation [88]. At a molecular level, master regulators for autophagy are mTOR and AMPK, both regulated by miR-155 and the miR-17-92 complex. Above, it was mentioned that miR-155 could repress activation of the RIP complex by inhibition with PTEN, as well as interfering with the Wnt/β-catenin and the Akt-pro survival pathway [29,67]. Additionally, the miR-17-92 complex results in the downregulation of mTOR [31]. FOXO3a, a pro-autophagitic factor, is negatively regulated by both miR-212 and miR-132. Over expression of miR-212 and miR-132 significantly disturbs autophagy and results in drastic cardiac hypertrophy and heart failure [69,89]. Additionally, the energy sensing pathway of AMPK can be blocked by disruption of the α1 subunit (AMPKα1), and activation of miR-148b directly inhibits its expression [90], thereby blocking the entire AMPK assembly.
Apoptosis, or programed cell death, is a process driven by cell death receptors. Cascading signals are mediated by many pro-and anti-apoptotic signals-caspases, the Bcl-2 family, and p53 [83,91]. Hypoxia-inducible apoptosis is a condition in which a low oxygen concentration induces the overexpression of HIF-1; this initializes apoptotic conditions by inducing high concentrations of BNIP3 and causing stabilization of p53 [92][93][94]. MiR-138 exhibits a protective effect against hypoxia-induced apoptosis via the MLK3/JNK/c-jun pathway [95]. By downregulating JNK, p38, Bax, and Caspase-3 levels, and upregulating Bcl-2, we can find an apoptotic for miR-320, namely IGF-1. Note that during inhibition of miR-320, the level of IGF1 mRNA is upregulated (Sun et al., 2017 [82]). Also, the anti-apoptotic signaling is the downregulation of miR-200c, as it increases levels of Bcl2 [96].
An important study by the American Heart Association and the American Stroke Association looked at cavernous malformations, which are best defined as circumscribed vascular lesions with thin-walled sinusoidal spaces lined with endothelial tissue and containing intravascular or intervascular calcifications [97]. Developmentally, these malformations control cardiac development via endothelial signaling of MEKK (MAPK/ERK pathway) and KLF [98]. PDCD10, a major role-playing factor in this condition, is heavily involved in cardiomyocyte autophagy. It has been shown to be susceptible to regulation by miR-613 by acting over LXRα [99], while PDCD4 is a direct target of miR-155 [100].
Yan et al. describe HF as a terminal stage of most types of cardiovascular diseases that always leads to a negative prognosis. Their study on the clinical relevance of using circulating levels of miR-423-5p as a potential biomarker found that the standard marker is B type natriuretic peptide (BNP) [101]. Similarly, BNP in MI, miR-208b, and miR-499 do not represent optimal biomarkers, although they are under great scrutiny [82]. Meanwhile, research is being conducted to use miR-1 and miR-30a, since they play crucial roles in cardiac hypertrophy and apoptosis by targeting key molecules in the signaling pathways [102]. Nonetheless, the current consensus opinion is that there are no well-established biomarkers for HF.

Future Directions
The clinical potential underlying miRs can be seen as a great area of opportunity, both for their targeting and as biomarkers. miRs themselves are clearly expressed in a tightly regulated fashion throughout development and during organ maintenance. At the same time, miRs show some peculiarities in expression during pathological altered states. In the clinic, knowing and understanding these imbalances takes us a step forward.
Important elements for the usage or target of miRs should be summarized, as certain elements can be considered strengths or weaknesses. Consider the size of a mature miR, which varies around 22 bp and can be feasibly seen as a target of an antagonizing sequence (antagomiR or anti-miR), analogous to the mRNA outcompeting it and thereby not inhibiting transcription [24,67,75]. The remarkable feature here is that a single upstream target can determine the fate of a whole signaling pathway instead of targeting individual factors by knockdowns or having to obliterate by fully knocking out a gene, which is not always a viable solution. Counterintuitively, pairing a 22 bp fragment is relatively easy, but we need to consider the dynamics of targeting a miR to its mRNA. This relates to the "seed", a 5 bp region at the 5 end and up to 2 more adjacent basepairs, as using the seed alone is often not sufficient for pairing. Therefore, it is not uncommon for non-canonical pairing to occur [1]. Hence, the potential for silencing off targets exists, so sequences need to be fully vetted by bioinformatics systems [23]. Another aspect to consider is that miRs have a short half-life, hence modifications or high quantities are considered as a possibility when intended for therapeutics. Moreover, this might lead to toxicity issues and alterations of biological properties [48]. Alternatively, a different strategy all together is blocking miRs that counter an undesired effect. To this extent numerous miRs inhibitors have been designed with different adjustments within their structure, in order to enhance their function. Some of the most common include 2 -MOE (2 -O-methoxyethyl) and 2 -fluoro, as they exhibit higher affinity when compared to others [103].
Remarkably in blood and other body fluids, miRNAs are stable (28 h-5 days) [103], in part due to the fact they are protected from RNA-degrading molecules by their association to lipoproteins in extracellular vesicles [4]. An interesting limitation for detection of miRs in body fluids stems from the use of the well-defined oligonucleotides used for PCR; if the reactions are not under the precise conditions required for small nucleic acids (including the design of the oligonucleotides), it can easily lead to a large number of artifacts [104]. Nevertheless, in a clinical setting, circulating miR-screening, due to its sensibility, can be an advantageous tool to pair with traditional testing, thereby providing a better framework for diagnosis. MiRs as serum biomarkers have been rigorously reviewed [74,79,101,104,105]. Well-known markers for MI, such as those mentioned by Sun et al., serve as a perfect example of well-defined markers circulating in the plasma. They found miR-1, miR-16, miR-21, miR-92a, miR-195, miR-208, miR-375, miR-494, miR-103, miR-107, miR-325, miR-499, and miR-874 to be upregulated during an before an MI, while at the same time miR-133a/b, miR-214, miR-873, miR-2861, miR-30b, miR-188-3p, and miR-145 are downregulated [82]. Moreover, we have mentioned above that both atherosclerosis and HF also (although not well established) have biomarkers, such as miR-21, miR-130a, miR-195, miR-92 (atherosclerosis) and miR-423-5p, miR-208a, miR-499, miR-16, miR-27a, miR-101, and miR-150 (HF) [78] (see Table S2).
Attempts to direct mimetics and antagomiRs to a specific organ pose a fascinating challenge. A direct injection the antagomiR to the organ is usually the best route, and delivery systems range from the use of liposome vesicles, polymers, and other viral particles. Yet, for the patient, it can lead to high costs [106]. The first set of experiments using antagomiR reported a systematic reduction of miRs. Intravenous administrations were carried out against miR-16, miR-122, miR-192, and miR-194, resulting in a notable decline of the resultant miRNA levels in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries, and adrenals [107]. Further studies have revealed the value of miRs to downregulate miRNAs in primates using locked nucleic acid (LNA) modification; these modifications connect the 2 -oxygen to the 4 -carbon of the ribose, resulting in a conformation which enhances mismatch discrimination [108]. The first set of around 20 clinical trials are currently underway using diverse delivery systems [17]. Nevertheless, the use of antagomiRs poses a number of potential setbacks, such as mistargets and delivery deficiencies. Yet, at the current speed of progress in science and technology, the opportunity to overcome them appears close. We expect the near future to hold many more key elements for personalized medicine.
Above, it was mentioned that miRs can have many potential targets. Consider, for example, the well-documented case of miR-29, which regulates fibrosis in the heart by targeting a whole set of components, such as elastins, fibrillins, and collagens-all components of the extra cellular matrix. Therefore, by using a well-designed antagomiR for miR-29, it has been possible to protect cardiomyocytes after damage from fibrotic remodeling [103]. Furthermore, during MI experiments it was found that antagomiRs for miR-92a and miR-320 reduced infarct size by contributing to recovery of blood vessels and reduction of apoptotic signals [57]. Meanwhile, Huang et al. considered the use of mesenchymal stem cells expressing miR-1. Elsewhere, it has been reported that miR-1 is sufficient to convert stem cell to cardiomyocytes. This experiment led to overall improvement of both cardiac function and a reduction in overall infarcted size [109]. Another set of experiments looked at miR-21. Dong et al. upregulated the expression of miR-21 by ischemic preconditioning-before MI-demonstrating considerable reduction in infarcted area. Moreover, the effect was even greater when injecting alongside miR-1 and miR-24 [57]. Experiments on atherosclerosis using antagomiRs (miR-33) showed a substantial elevation in high-density lipoprotein cholesterol (HDL) and reduction in very low-density lipoprotein cholesterol (VLDL). Additionally, LNA-AntagomiR (miR-122) showed promise in cholesterol reduction, while 2 -O-methoxyethyl phosphorothioate (miR-122) enhanced liver steatosis [104]. AntagomiR (miR-145) has been used in rodents to promote a reduction of plaque size and enhance vasculature. The results showed a marked reduction in plaque in aortic sinuses and the necrotic core, and increased collagen, which promoted contractile VSMC [74,104]. These studies demonstrated how, using an antagomiR or a miR-mimic, it is possible to achieve a desired outcome.
Since their initial discovery, small non-coding RNAs, have played an instrumental role in deciphering the nature and mechanics of human biology and its conditions. As we have seen in this overview of CVDs, they are instrumental both in the way they orchestrate through positive and negative feedback, and direct control. Ultimately, we see that they work in groups, many belonging to a family or super family of miRs. In the right balance, they are responsible for the correct cardiac environment. We can look forward to the next few years, in which technological advances will take us further in understanding and uncovering more insights into miRs, mimics, and antagomiRs. It is very likely that their localized use will be a common method of personalized treatment for many illnesses. In addition, we expect an even faster uptake in the number of clinics using screening methods to identify miR levels in patients as a way to easily access disease information. Funding: Institutional funding provided by Tecnologico de Monterrey.

Conflicts of Interest:
The authors declare no conflict of interest.