MicroRNAs Regulating Renin–Angiotensin–Aldosterone System, Sympathetic Nervous System and Left Ventricular Hypertrophy in Systemic Arterial Hypertension

MicroRNAs are small non-coding RNAs that regulate gene and protein expression. MicroRNAs also regulate several cellular processes such as proliferation, differentiation, cell cycle, apoptosis, among others. In this context, they play important roles in the human body and in the pathogenesis of diseases such as cancer, diabetes, obesity and hypertension. In hypertension, microRNAs act on the renin–angiotensin–aldosterone system, sympathetic nervous system and left ventricular hypertrophy, however the signaling pathways that interact in these processes and are regulated by microRNAs inducing hypertension and the worsening of the disease still need to be elucidated. Thus, the aim of this review is to analyze the pattern of expression of microRNAs in these processes and the possible associated signaling pathways.


Introduction
Systemic arterial hypertension (SAH) is a multifactorial disease that is associated with genetic factors, such as inherited genes that favor increased blood pressure and environmental factors, including hypersodic and hypercaloric diet, overweight and obesity, physical inactivity, alcohol consumption in excess, advanced age, among other factors [1]. SAH is also defined as a chronic state of high blood pressure levels [2].
It is estimated that the global prevalence of SAH is greater than 30% in individuals aged 20 years or more. This rate varies when analyzed separately in some countries, with the percentage difference being almost 5% higher in medium-and low-income economy countries when compared to high-income economy countries [3]. In addition, it is known that the SAH is also associated to the development of type 2 diabetes and other comorbidities.
Several processes are associated with increased blood pressure levels and later with SAH, including: changes in the renin-angiotensin-aldosterone system (RAAS), activation of the sympathetic nervous system (SNS), endothelial dysfunction and increased oxidative stress [4]. These long-term pathophysiological processes result in hemodynamic pressure overload promoting cardiac remodeling and pathological cardiac hypertrophy in SAH [5].
Little is known, however, about the molecular mechanisms that initiate and govern these pathophysiological processes in SAH, which begin to be elucidated through microR-NAs (miRNAs). MiRNAs are small ribonucleic acids (RNAs), non-coding RNA and gene expression regulators at the post-transcriptional level [6], their function is to degrade or inhibit one or several target messenger RNAs [7,8].
Some studies have reported miRNA deregulation in SAH, promoting activation of RAAS and SNS [9], and inducing pathological cardiac hypertrophy of the left ventricle [10]. However, the molecular mechanisms regulated by miRNAs that regulate the RAAS, SNS and left ventricular hypertrophy (LVH) in SAH still need to be elucidated. Thus, the aim of this review is to analyze the expression of deregulated miRNAs in RAAS, SNS and LVH in SAH, including possible signaling pathways associated with these processes.

MiRNAs and Activation of the Renin-Angiotensin-Aldosterone System in SAH
The renin-angiotensin-aldosterone system (RAAS) is of fundamental importance in the control of extravascular volume and blood pressure [11]. Due to its regulatory role, RAAS inhibition has therapeutic implications for several chronic diseases, such as SAH, type 2 diabetes and heart failure [12].
Renin is an enzyme synthesized by the juxtaglomerular cells of the kidney and stored in its inactive form as prorenin [13]. In situations of blood pressure drop, circulating volume depletion, reduced serum sodium concentration and sympathetic activation, prorenin molecules will be cleaved and renin will be released, which will act as the main regulator of RAAS [14].
Angiotensinogen is a circulating protein, synthesized by the liver, which will be metabolized under the action of renin, forming angiotensin I, a peptide. The conversion of angiotensin I (Ang-I) to angiotensin II (Ang-II) occurs mainly through the action of the angiotensin-converting enzyme (ACE) in the pulmonary microcirculation [15].
Angiotensin II acts by stimulating the type 1 angiotensin II (AT1R) receptor, causing vasoconstriction in the cardiovascular system, reducing the excretion of sodium and water, stimulating the synthesis of aldosterone, a mineralocorticoid synthesized in the glomerulosa of the adrenal cortex, among other functions [12,16]. Aldosterone also modulates circulating volume through marked stimulation of sodium reabsorption at the level of the renal tubules, with consequent fluid retention, in addition to potassium excretion [14,16].
RAAS imbalances contribute to the development of SAH, due to frequent and intense vasoconstriction of arterioles that cause an increase in peripheral vascular resistance and subsequently in blood pressure, in the long term, target organ damage occurs with more serious consequences [11,17].
MiR-483 targets multiple RAAS genes, such as angiotensinogen (AGT), ACE-1, ACE-2 and angiotensinogen receptor 2 (AGTR2), however, the expression of miR-483 is downregulated in vascular smooth muscle cells (VSMCs) stimulated with Ang-II, which leads to overexpression of its target genes and vascular hypertrophy [21]. MiR-143/145 cluster also regulates ACE-1, and when these miRNAs are poorly expressed in VSMCs, it promotes an increase in ACE-1 expression, inducing a reduction in its contractile phenotype and vascular dysfunction [22].
In this context, VSMCs stimulated with Ang-II present reduced miR-155 expression and increased AT1R expression, leading to increased cell proliferation, however, the increase in the expression of this miRNA can abrogate this effect [23]. Likewise, another miRNA, miR-130, was overexpressed, reducing the expression of the target gene GAX, contributing to the proliferation of VSMCs when stimulated with Ang-II [24]. MiR-27a also had its expression elevated in VSMCs stimulated with Ang-II, reducing the expression of α-smooth muscle-actin (α-SMA), leading to proliferation and migration of VSMCs, promoting change in contractile phenotype and reduction in vascular function [25], which favors an increase in peripheral vascular resistance and consequently induces an increase in blood pressure.
In another study with Ang-II-stimulated cardiac fibroblasts and HEK293N cells, four miRNAs were found to be overexpressed: miR-29b, miR-129, miR-132 and miR-212, activating Gαq/11, the extracellular regulated kinase1/2 (ERK-1/2) and AT1R, and the activation of this pathway can promote increased blood pressure [29]. MiR-132/212 cluster is overexpressed in the heart, aorta, kidney and blood circulation of Ang-II-induced hypertensive rats, as well as in the arteries of hypertensive patients, inducing endothelin receptor activation and cardiac hypertrophy [30]. It has also been shown that miR-132 regulates plasma renin levels by modulating blood pressure [31].
In hypertensive rats, miR-16 is overexpressed, reducing the expression of the vascular endothelial growth factor (VEGF), promoting an anti-angiogenic effect, and miR-21 is also highly expressed decreasing the expression of the anti-apoptotic Bcl-2, inducing apoptosis. On the other hand, in this same study, miR-126 is downregulated by increasing the expression of the phosphoinositol-3 kinase regulatory subunit 2 (PI3KR2) which negatively modulates the VEGFR, mitogen activated protein kinase (MAPK) and phosphoinositol-3 kinase (PI3K) signaling pathways, generating microvascular rarefaction in hypertension [32].
Microvascular rarefaction is a process that occurs in individuals with SAH that decreases blood flow in skeletal muscle capillaries, inducing an increase in total peripheral resistance, generating an increase in blood pressure. This process promotes changes in the expression of several miRNAs that regulate vascular remodeling in SAH, being an important factor to be analyzed in future studies for reverse modulation of these miRNAs in an attempt to attenuate this process [33].
Clinical studies also show changes in the expression of miRNAs in hypertensive patients. MiR-136 is downregulated in the serum of patients with hypertension and is associated with elevated levels of RAAS biochemical markers [34]. However, miR-202 is overexpressed in the blood of hypertensive patients, by reducing the receptor soluble (sST2), being associated with high levels of Ang-II and vascular inflammation [35].
Other miRNAs are also with high expression in hypertensive patients, such as miR-21, miR-126, miR-196a and miR-451, while others are with reduced expression, such as miR-181a, miR-638 and miR-663. It was even evidenced that miR-181a and miR-663 target the renin gene, demonstrating that these miRNAs potentially regulate blood pressure [36].
Another miRNA that regulates the renin gene is miR-25. This miRNA is downregulated in the serum of hypertensive patients, elevating the renin expression, promoting RAAS activation and consequently leading to hypertensive heart disease [37]. MiR-155 is also downregulated in the plasma of hypertensive patients, increasing AT1R expression, activating the RAAS signaling pathway, increasing blood pressure [38] (Table 1). ↓ miR-483-3p VSMCs-human and rat aortic smooth muscle cells miR483-3p is reduced after in vitro stimulation with angiotensin II, which activates the renin angiotensin aldosterone system (RAAS).
Multiple components of the RAS: ACE1, ACE2, AGTR2 [21] ↑ miR-130a VSMCs were prepared from the thoracic aorta of Sprague-Dawley rats miR-130a induces the proliferation of VSMCs, by targeting GAX, which has inhibitory actions on VSMCs proliferation.

Cardiac fibroblasts treated by AngII
A group of dysregulated miRNAs when treated with AngII, demonstrating important roles in hypertension and cardiac fibrosis.

IKBKE
[42] TGF-β1 [65] Thus, the RAAS signaling pathway is modulated by miRNAs to regulate blood pressure, however, many other miRNAs can regulate these same RAAS genes in SAH, but further studies are needed to confirm this hypothesis.

MiRNAs and Sympathetic Nervous System Activation in SAH
The sympathetic nervous system (SNS) plays a fundamental role in blood pressure control through regulatory mechanisms expressed based on the release of neurotransmitters (epinephrine, norepinephrine and dopamine) that act on vessels, kidneys and heart. Thus, changes in the functions of this system are related to the development of cardiovascular disorders [66].
Cardiac output and systemic vascular resistance are the main target components for the action of sympathetic neurotransmitters through their release, by central and reflex mechanisms, and binding to their adrenergic (α and β) and dopaminergic receptors. These mechanisms, when activated, promote an arteriolar vasoconstriction (through α-receptors) and an increase in cardiac output (through β-receptors) resulting in an elevation of blood pressure [67,68].
Activation of the SNS occurs in states of physical or emotional stress. However, in early stages of SAH, known as a hyperkinetic circulatory state, there is an increase in adrenergic impulse and a decrease in parasympathetic function [67], that is, there is an increase in plasma levels of sympathetic neurotransmitters concomitant with a loss of vagal inhibitory function. Furthermore, the magnitude of sympathetic activation parallels the severity of blood pressure elevation [68].
MiRNAs also participate in the regulation of the SNS. Specifically, miR-181a was downregulated in a genetic model of massive SNS activation, leading to increased renin expression, RAAS activation, and consequently hypertension in mice [49]. Overexpression of miR-135a and miR-376 were also associated with increased sympathetic nerve activity, contributing to exacerbated blood pressure and inflammation in spontaneously hypertensive rats [44].
In this context, miR-22 is also highly expressed in spontaneously hypertensive rats, reducing the expression of the Chromogranin A (CHGA), inducing greater central and peripheral nerve activity, contributing to the elevation of blood pressure [51]. A polymorphism in the CHGA 3'-untranslated region known as C+87T (rs7610), promotes increased inhibition of CHGA by miR-107, leading to increased sympathetic nerve activity, autonomic dysregulation, increased blood pressure and renal disease in a hypertensive mouse model [45].
Renal disease in hypertensive patients is very common due to increased renal sympathetic nervous system and this hyperactivation induces reduced expression of miR-133a [60]. This same study showed that the renal sympathetic denervation in these patients promotes increased expression of miR-133a, attenuating blood pressure, including being associated with decreased risk of developing hypertensive heart disease [60].
Other miRNAs, such as miR-200a, miR-200b, miR-205, miR-141, miR-192 and miR-429, are overexpressed in hypertensive patients with renal disease leading to nephrosclerosis, and the degree of elevation of these miRNAs was correlated with disease severity [69].
Furthermore, other miRNAs can be dysregulated due to activation of the sympathetic nervous system and inhibition of the parasympathetic nervous system in SAH, favoring the hemodynamic overload that generates long-term left ventricular hypertrophy.

MiRNAs and Left Ventricular Hypertrophy in SAH
Under physiological conditions, stroke volume is regulated by preload through mechanisms that involve the extension of cardiac fibers at the end of diastole associated with resistance imposed by afterload [70]. Mechanical stress on the heart induces changes involving strain (related to increased afterload) and shear stress (related to blood friction against the vessel wall) resulting in compensatory adaptive effects when chronically altered in order to keep the cardiac output [71].
This mechanical stress is one of the triggering factors of cardiac remodeling, which occurs through processes of cardiomyocyte hypertrophy, hyperplasia, hypertrophy of nonmuscle cells and interstitial proliferation [72]. Cardiac cell remodeling and hypertrophy, in response to mechanical stress in SAH, are the mechanisms that lead to left ventricular hypertrophy (LVH) [73]. LVH in SAH induces an increase in muscle mass and myofibril growth in parallel, generating concentric hypertrophy, reducing the internal area of the cardiac chamber. Hyperplasia of vascular structures and collagen accumulation also occur, favoring cardiac fibrosis [74].
On the other hand, Ang-II-induced pressure overload in rats reduced the expression of miR-19a and miR-19b, increasing the expression of phosphodiesterase 5A (PDE5A), generating LVH, including, as the authors show in the same study, that a model of transgenic mouse overexpressing miR-19a and miR-19b reduced PDE5A expression, decreasing cardiac hypertrophy [55], demonstrating that an expression pattern response of miRNAs in cell culture can be quite different from the response in animal models.
MiR-21, miR-126 and miR-146 are overexpressed in a C576BJ mouse model of cardiac hypertrophy, while miR-29b, miR-133a, miR-133b, miR-149, miR-150 and miR-185 were downregulated after aortic binding constriction [46]. MiR-132 and miR-212 are also overexpressed, decreasing the expression of the FoxO3 transcription factor, inducing attenuation of autophagy and activation of the pro-hypertrophic calcineurin/NFAT signaling pathway, generating LVH, in both, in the culture of primary cardiomyocytes with different hypertrophic stimuli and in mice with transaortic constriction (TAC) and cardiac hypertrophy [40]. Likewise, in both in vitro and in vivo models stimulated with Ang-II, the expression of miR-410 and miR-495 is increased, modulating the expression of the hypertrophic genes Nppa and Nppb, promoting a robust hypertrophy of cardiomyocytes [48].
In an model of hypertensive mice overexpressing renin, it promoted SAH by increasing the expression of miR-208a and 208b, decreasing the expression of the transcription factor SOX-6, which is a repressor of alpha-myosin heavy chain (MyHC), increasing the expression of the latter gene, inducing LVH [47].
MiR-208b was also overexpressed in peripheral blood mononuclear cells from patients with SAH, in addition, other miRNAs were also overexpressed in these samples, such as miR-1, miR-21 and miR-499, while miR-26b and miR-133a were with reduced expression and this expression pattern correlated with LVH in these patients [58]. In studies in humans, animals and cells, the reduction of miR-133a expression promotes LVH or cardiomyocytes hypertrophy and the overexpression of this miRNA induces an anti-hypertrophic effect [75,76].
The miR-29 family (miR-29a, miR-29b and miR-29c), in turn, is overexpressed in the plasma of patients with SAH, regulating genes of the fibrotic and hypertrophic process, having a strong correlation with high blood pressure and LVH [61]. In agreement with these results, miR-29a was also shown with increased expression in the plasma of hypertensive patients with positive association with LVH [77].
On the other hand, miR-29b had its expression reduced in a mouse model of Ang-II-induced hypertension, increasing the expression of cholagen-1 (COL-I), transforming growth factor-β (TGF-β), α -SMA, tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), with reduced expression of the mothers against decapentaplegic homolog 7 (SMAD7) gene, inducing LVH. However, the induction of SAMD7 overexpression prevented the loss of miR-29b, decreasing the expression of COL-I, TGF-β, α-SMA, TNF-α, IL-1β, reducing the inflammatory and fibrotic profile, in addition to attenuating the heart damage and LVH in these animals [54]. This finding demonstrates that the expression pattern of miRNAs may be different in hypertensive patients compared to animal models.
Another miRNA that is associated with the process of inflammation and cardiac hypertrophy is miR-155. In both in vivo and in vitro models of Ang-II-induced cardiac hypertrophy, miR-155 is overexpressed, reducing the expression of the inhibitor of nuclear factor kappa-B kinase subunit epsilon (IKBKE), promoting activation of the nuclear factor kappa-B (NF-κB), inflammation and cardiac hypertrophy [42]. In this same work, it was shown that the overexpression of the long non-coding RNA known as cytoskeleton regulator RNA (CYTOR), can serve as a sponge for miR-155, reducing the expression of this miRNA, and consecutively increasing the expression of IKBKE, inhibiting the NF-κB pathway, reducing inflammation and cardiomyocyte hypertrophy [42].
MiR-155 also had increased expression in the plasma of hypertensive patients, and this high expression was associated with inflammatory markers such as interleukin-6 (IL-6) and c-reactive protein [65]. Other circulating miRNAs also dysregulated in the plasma of hypertensive patients, such as miR-29 and 30a, are overexpressed, whereas miR-133 is downregulated [64], and these miRNAs are associated with the processes of inflammation and LVH [75,78].
Many other miRNAs are regulating genes and signaling pathways in SAH, including other non-coding RNAs; the main miRNAs that regulate signaling pathways in the RAAS, SNS and LVH are shown in a schematic representation in (Figure 1). Furthermore, other epigenetic processes, such as DNA methylation, histone acetylation and deacetylation, are also being modulated by changing transcriptomics during the pressure overload process promoting SAH. and LVH [75,78].
Many other miRNAs are regulating genes and signaling pathways in SAH, including other non-coding RNAs; the main miRNAs that regulate signaling pathways in the RAAS, SNS and LVH are shown in a schematic representation in (Figure 1). Furthermore, other epigenetic processes, such as DNA methylation, histone acetylation and deacetylation, are also being modulated by changing transcriptomics during the pressure overload process promoting SAH.

Overlapping miRNAs in RAAS, SNS and LVH in SAH
After reviewing the expression of miRNAs in RAAS, SNS and LVH in in vitro, in vivo and clinical studies, despite different methodologies applied in these works, it was possible to identify six miRNAs (miR-21 miR-155 miR-132 miR-29b miR-126 miR-212) with altered expression regulating RAAS and LVH processes. It was also possible to identify two miRNAs (miR-181a miR-135a) modulating the RAAS and SNS processes. No miRNA was identified associated with the SNS and LVH processes. However, one miRNA was observed to regulate all processes, the miR-133a ( Figure 2).
MiR-133a plays an important role in cardiac development and is known to regulate some cardiovascular diseases [79][80][81]. Here it was identified that miR-133a is involved in the regulation of three processes that are fundamental for the development of SAH and the clinical worsening of the disease. In this context, miR-133a is downregulated regulating signaling pathways in RAAS, SNS and LVH, as previously described. Interestingly, it was identified that miR-133a is regulating the PRR gene, activating the RAAS and SNS, promoting LVH, aggravating the disease. In addition, miR-133a is associated with other signaling pathways such as β-MHC and atrial natriuretic factor (ANF). Thick red upward arrow: upregulation of miRNA or target gene; thick green downward arrow: downregulation of miRNA or target gene; blue thin arrow: indicates that miRNA regulates that gene; red thin arrow: indicates that the change in the signaling pathway promotes a specific outcome.

Overlapping miRNAs in RAAS, SNS and LVH in SAH
After reviewing the expression of miRNAs in RAAS, SNS and LVH in in vitro, in vivo and clinical studies, despite different methodologies applied in these works, it was possible to identify six miRNAs (miR-21 miR-155 miR-132 miR-29b miR-126 miR-212) with altered expression regulating RAAS and LVH processes. It was also possible to identify two miRNAs (miR-181a miR-135a) modulating the RAAS and SNS processes. No miRNA was identified associated with the SNS and LVH processes. However, one miRNA was observed to regulate all processes, the miR-133a ( Figure 2).
MiR-133a plays an important role in cardiac development and is known to regulate some cardiovascular diseases [79][80][81]. Here it was identified that miR-133a is involved in the regulation of three processes that are fundamental for the development of SAH and the clinical worsening of the disease. In this context, miR-133a is downregulated regulating signaling pathways in RAAS, SNS and LVH, as previously described. Interestingly, it was identified that miR-133a is regulating the PRR gene, activating the RAAS and SNS, promoting LVH, aggravating the disease. In addition, miR-133a is associated with other signaling pathways such as β-MHC and atrial natriuretic factor (ANF).
In addition to regulating these three overlapping processes, miR-133a also regulates vascular remodeling and fibrosis through the membrane type-1 matrix metalloproteinase (MT-1 MMP) target gene. MiR-133a was also reduced in aortic fibroblasts exposed to biaxial cyclic stretch, generating tension and vascular remodeling, including being downregulated in thoracic aortic tissue of Ang-II-induced hypertensive mice and in spontaneously hypertensive mice [82].
In situations of myocardial ischemia that occur in many hypertensive patients, miR-133a is also downregulated, promoting an increase in MT-1 MMP, in addition to elevating other pro-fibrotic genes such as transforming growth factor beta receptor 1 (TGFBR1), latent transforming growth factor binding protein 1 (LTBP1), matrix metalloproteinase 9 (MMP9), phosphorylated Smad2 (pSMAD2) and COLIAI, inducing activation of TGF-β signaling pathway, leading to vascular and cardiac fibrosis [83]. In addition to regulating these three overlapping processes, miR-133a also regulates vascular remodeling and fibrosis through the membrane type-1 matrix metalloproteinase (MT-1 MMP) target gene. MiR-133a was also reduced in aortic fibroblasts exposed to biaxial cyclic stretch, generating tension and vascular remodeling, including being downregulated in thoracic aortic tissue of Ang-II-induced hypertensive mice and in spontaneously hypertensive mice [82].
In situations of myocardial ischemia that occur in many hypertensive patients, miR-133a is also downregulated, promoting an increase in MT-1 MMP, in addition to elevating other pro-fibrotic genes such as transforming growth factor beta receptor 1 (TGFBR1), latent transforming growth factor binding protein 1 (LTBP1), matrix metalloproteinase 9 (MMP9), phosphorylated Smad2 (pSMAD2) and COLIAI, inducing activation of TGF-β signaling pathway, leading to vascular and cardiac fibrosis [83].
Furthermore, miR-133a was shown to be reduced in the left ventricle of rats with chronic administration of AngII, increasing the expression of the COLIAI gene, generating myocardial fibrosis [84]. All this evidence demonstrates that miR-133a plays a critical role in SAH, acting as a key element in this disease.

Conclusions
Several miRNAs are regulating target genes and altering signaling pathways in RAAS, SNS and LVH in SAH. Some of these miRNAs are associated and interact to activate these systems simultaneously and worsen the clinical status of these patients. Specifically, miR-133a has a key multiregulatory role in the three analyzed processes, regulating the PRR signaling pathway, and is also associated with other deleterious situations in SAH, such as cardiac fibrosis and myocardial ischemia. Thus, further studies are needed to analyze the expression of miRNAs in RAAS, SNS and LVH, and especially the activation of miR-133a as a potential therapy for inactivation of RAAS and SNS, inducing possible LVH attenuation in SAH. Furthermore, miR-133a was shown to be reduced in the left ventricle of rats with chronic administration of AngII, increasing the expression of the COLIAI gene, generating myocardial fibrosis [84]. All this evidence demonstrates that miR-133a plays a critical role in SAH, acting as a key element in this disease.

Conclusions
Several miRNAs are regulating target genes and altering signaling pathways in RAAS, SNS and LVH in SAH. Some of these miRNAs are associated and interact to activate these systems simultaneously and worsen the clinical status of these patients. Specifically, miR-133a has a key multiregulatory role in the three analyzed processes, regulating the PRR signaling pathway, and is also associated with other deleterious situations in SAH, such as cardiac fibrosis and myocardial ischemia. Thus, further studies are needed to analyze the expression of miRNAs in RAAS, SNS and LVH, and especially the activation of miR-133a as a potential therapy for inactivation of RAAS and SNS, inducing possible LVH attenuation in SAH.