Epigenetics and Arterial Hypertension

Chapter 7

Epigenetics and Arterial Hypertension: Evidences and Perspectives S. Friso, C.A. Carvajal, F. Pizzolo, C.E. Fardella and O. Olivieri

ABBREVIATIONS AH 5hmCyt 5mCyt PBMCs

arterial hypertension 5-hydroxymethylcytosine 5-methylcytosine peripheral blood mononuclear cells

Key Concepts 1. The current knowledge on the link between epigenetic marks and AH is promising but yet to be deepen, potentially leading toward novel implications in AH preventive and therapeutic strategies. 2. Epigenetic changes in DNA refer to DNA methylation and hydroxymethylation, posttranslational regulation by histone modifications, and noncoding RNAs. 3. Differently from genetic features of DNA, epigenetic changes are potentially reversible by environmental/nutritional factors, and therefore of special interest for multifactorial diseases such as arterial hypertension (AH).

INTRODUCTION Arterial Hypertension and Epigenetics Arterial hypertension (AH) is a multifactorial disease, in which a complex pathogenesis involves several mechanisms and endocrine
metabolic systems. Differential etiologies of AH are known to take place in the dynamic interplay among genetic factors and the diverse environmental backgrounds leading to changes in multiple biological pathways that eventually trigger the genesis of Translating Epigenetics to the Clinic. © 2017 Elsevier Inc. All rights reserved.

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this complex disorder that primarily involves the cardiovascular system but can certainly be considered a chronic systemic disease [1
3]. AH is indeed a major risk factor for cardiovascular diseases such as stroke, myocardial infarction, heart failure, and end-stage renal disease. A large portion of the worldwide population suffers from AH, which gives to this disease the rank of one of the major concerns for public health issues, especially in Western countries [4
9]. To unveil in more depths the pathogenetic routes that stand behind the development of hypertension is, therefore, crucially important to outline efficacious modalities to prevent and treat such a widespread illness [9,10]. Since a positive family history for AH is a well-known major risk factor for the development of the disease, several studies have focused, in the latest years, on examining the genome sequence of people affected by AH to find genetically driven molecular mechanisms that may alter a certain pathway or another by using either a candidate gene or a genome-wide association study approach [1,3,11]. Considering that pathogenetic mechanisms related to kidney function and those related to the function of endocrine systems are the most frequently implied in a number of forms of AH, most studied precisely focused on forms of AH with a genetic background [12
15]. Latest genome-wide association studies greatly contributed to the advancement of the comprehension of the part played by the genetic components in hypertensive disease [16
20]. It has become clear, however, that a mere DNA-sequence-based approach may only partially explain the contribution of the genome structure and function in the pathogenesis of hypertension that should instead be considered from a broader point of view which involves epigenetic mechanisms that, despite their heritability, are potentially modifiable by environmental factors [21]. Precisely for their function in gene expression regulation, several phenomena that regulate gene expression through epigenetics are lately emerging as a possible step forward in the understanding of AH development [22] and opened up to novel and more complex role of gene functions that are not merely due to the genome sequence and structure by themselves but to the way by which a specific gene is regulated by heritable mechanisms in disease pathogenesis [21,23]. Epigenetic mechanisms refer precisely to phenomena that are implicated in gene expression regulatory systems that do not involve changes in the DNA sequence or copy number variation but occur over the mere nucleotide sequence of the genome [22]. Most of the studies aimed at evaluating the role of epigenetics in AH in humans have focused their interest on DNA methylation, both global [24] and gene-specific, mostly by a gene-target approach [25] while emerging evidence shows a function in AH pathogenesis and progression also for other epigenetic phenomena such as histone modifications and noncoding RNAs [26,27]. This chapter aims at giving an overview of the current knowledge of the importance of epigenetic mechanisms in AH by considering the current available observations from cell culture, animal and human studies.

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EPIGENETIC REGULATION THROUGH DNA METHYLATION, HISTONE MODIFICATION, AND MICRORNA Global DNA Methylation in Hypertension Epigenetic modifications consist in molecular processes that regulate gene expression and genome stability and specifically refer to nucleic acids methylation and hydroxymethylation, histone modifications, nucleosome positioning, and posttranscriptional gene regulatory phenomena influenced by noncoding RNAs and RNA-binding proteins (Fig. 7.1) [22,28,29]. Among the epigenetic features of DNA, methylation at the cytosine
guanine (CpG) sequence sites is, so far, considered the major and certainly the most studied

FIGURE 7.1 Main epigenetic marks in eukaryotic cells. DNA methylation, the covalent binding of a methyl group to the 50 carbon of cytosine at CpG dinucleotide sequences, usually inhibits gene transcription if occurring in the gene promoter but it may also promote transcription when localized at a specific gene exons sites. Posttranslational histone modifications are epigenetic phenomena that involve histone tail residues and influence gene expression by controlling the dynamics of chromatin. Histone tail methylation and acetylation are reported as the main histone epigenetic marks at histone sites as schematically shown in the upper right panel of the figure. The biogenesis of noncoding RNAs including small noncoding RNAs, i.e., miRNAs, occurs across the cell nucleus and cytoplasm; as schematically shown in the figure, the cytoplasm is the place where mature miRNAs exert their downregulating effect on gene transcription by leading to target mRNA degradation or by mRNA translational repression.

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epigenetic feature in mammalian DNA (Fig. 7.1) [30]. It consists in the covalent binding of a methyl group to the 50 position of a cytosine that mainly occurs within the CpG dinucleotide sequences at promoter or first exons regions [22,31
33]. The availability of methyl groups for biological methylation reactions depends on the amount of the universal methyl-donor, S-adenosylmethionine (SAdoMet), that is necessary for the function of specific DNA methyltransferases (DNMTs) including those acting at the level of DNA, RNA as well as those methylating functions occurring at histone tail sites [29]. The endproduct of those enzymatic reactions catalyzed by DNMTs is S-adenosylhomocysteine (SAdoHcy) which is not only the final product but also a strong inhibitor of the methyltransferase enzymes activity. Both SAdoMet and SAdoHcy availability are controlled within the folate-dependent one-carbon metabolism, hence establishing a strict link between the function of biological methylation and nutritional factors, therefore providing a paradigmatic example for the close relationship between epigenetics and environment modulating the multifaceted epigenetic patterns in complex diseases [34]. Both an increased DNA methylation and a lower content of 5-methylcytosine (5mCyt) have been described in many diseases, where a reduced global content of 5mCyt, usually related to genome instability, may correspond to an increased methylation status at specific gene loci, as often documented in several neoplastic diseases [35,36]. The importance of DNA methylation has been only recently highlighted as a possible mechanism affecting pathologies other than cancer, among which cardiovascular diseases comprising AH [37
40]. There is, therefore, a conspicuous interest toward the investigation of the role of specific epigenetic mechanisms in the pathogenesis of composite diseases such as those affecting the cardiovascular system including AH particularly considering the role of environmental
nutritional factors (i.e., salt intake, as a pathognomonic example) in affecting their phenotypic expression precisely through epigenetics [24,26,40
42]. The methylation of DNA determines the regulation of gene expression by modulating the binding with transcriptional protein complexes in order to direct the gene regulatory information to define which portions of the genome are either functioning or silent [43,44]. DNA methylation is heritable and can be transmitted through cell divisions to maintain the proper cell function and it can be defined as either global or gene-specific depending on whether it is considered the overall content of 5mCyt in the genome or the DNA methylation pattern at a specific gene site [33]. Functions of DNA methylation are mainly those of maintaining genome stability at the global level and to regulate more specifically when and how a certain gene should be active or inactive at a gene-specific level [36]. There are only few reports, so far, on DNA methylation pattern and its implication in AH even though the interest toward the role of DNA

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methylation in AH is rapidly gaining significance [26,40]. An overview of the main epigenetic modifications in AH, including those pertaining to DNA methylation, is shown in Table 7.1 and Fig. 7.2. A reduced content of 5mCyt in PBMCs DNA of patients affected by essential hypertension as compared to a control group has been reported [24]. Nevertheless, one of the main issues in the interpretation of these findings is to establish the value of DNA methylation status when measured in PBMCs in hypertensive patients, especially if one considers the tissue and cell specificity of the epigenetic phenomena and that the meaning of an epigenetic alteration at PBMCs molecular levels is not immediately evident if considering the pathogenesis of hypertensive disease. Undeniably, it is quite intriguing, at the same time, to contemplate the possibility of defining the epigenetic signatures from an easily available tissue such as PBMCs in hypertensive individuals or in persons at risk for the disease for possible preventive perspectives. It should be also noted that an increasing number of reports show a probable role as a disease marker of methylation measured in PBMCs DNA even in cancer [55,56]. It may be then speculated that the metabolic pathways regulating methylation of DNA may be altered in the entire organism and be therefore reflected in the alteration of DNA methylation in blood cells, even in AH [56]. A precise and highly reproducible method could be crucial, in this regard, to detect minimal differences in DNA methylation in human studies especially if the determination of DNA methylation is thought as a possible disease biomarker [55,57,58].

DNA Hydroxymethylation and Hypertension Besides 5mCyt, another modified base, 5hmCyt, appears to have a main function in gene expression regulation [59,60]. 5hmCyt is an intermediate compound in the 5mCyt demethylation reaction catalyzed by the ten-eleven translocation family of methylcytosine dioxygenases that regulate the maintenance of patterns of methylation for gene expression regulation and development processes. Valinluck et al. [61] reported that 5hmC at CpG sites was found to inhibit the binding of methyl-CpG binding protein 2, which is a transcriptional repressor, suggesting a potential role of 5hmC in gene expression regulation. The precise function of 5hmCyt in gene expression as well as its relationship to pathogenesis of diseases, including AH, is unclear in humans although some evidences have been shown in a rodent model [45]. In an in vivo model of hypertensive disease using a Dahl salt-sensitive (SS) rat model (Table 7.1), both 5mCyt and 5hmCyt were studied at single-base resolution by analyzing the renal outer medulla [45]. The maps showed that, in response to salt intake, the methylation patterns significantly changed where the lower 5mCyt content was associated with increased gene expression and the higher 5hmCyt was usually associated with higher gene expression, consistent with the notion that the increase of 5mCyt generally

TABLE 7.1 Global Methylation and Hydroxymethylation and Gene-Specific Methylation Patterns in AH Epigenetic Phenomenon

Epi-Mark

Status

Sample Source

Affected Function Related to AH

Reference

Global DNA methylation

5mCyt

Low

Human PBMCs

Genome instability

[23]

DNA hydroxymethylation

5hmCyt

High

Dahl SS rat renal medulla cells

Higher gene expression

[45]

Gene-specific DNA methylation

Gene

NCBI ID

MIM

HSD11B2

NG_016549

614232

5mCyt

High

Human PBMCs

Renal sodium balance

[25,46,47]

NKCC1 (SLC12A2)

NM_001046

600840

5mCyt

High

SHR

Ionic transport (ubiquitous)

[48
50]

sACE

NG_011648

106180

5mCyt

High

Human PBMCs; cell culture (HepG2, HT29, HMEC-1, SUT)

RAAS

[51]

AGT

NG_008836

106150

5mCyt

Low

Cultured human cells

RAAS

[52]

ADD1

NG_012037

102680

5mCyt

Low

Human PBMCs

Modulation of ionic pumps and transporters (ubiquitous)a

[53]

ESR1 (ER-alpha)

NG_008493

133430

5mCyt

High

Ovine uterine arteries

Gestation (hypoxia)

[54]

Epi-Mark, epigenetic marker; HepG2, liver epithelial carcinoma cells; HMEC-1, human microvascular endothelial cells; HT29, colon epithelial adenocarcinoma cells; 5hmCyt, 5-hydroxymethylcytosine; PBMCs, peripheral blood mononuclear cells; RAAS, renin
angiotensin
aldosterone system; SHR, salt-hypertensive rat; SUT, lung epithelial adenocarcinoma. a ADD1 gene with gender dimorphism.

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FIGURE 7.2 Epigenetic modifications in specific genes related to AH. DNA Methylation: A high CpG methylation in HSD11B2 promoter decreases its expression, which could impair the renal metabolism of cortisol to cortisone. Cortisol can activate mineralocorticoid receptor (MR) and increase the renal sodium reabsorption and induce AH. Inversely, NKCC1 gene expression is also affected by CpG promoter methylation. Low CpG promoter methylation in NKCC1 - in both endothelial cells (ECs) and in smooth muscle cells (SMCs)- increases vasoconstriction. Histone Modification: Specific histone deacetylation of WNK4 promoter in an nGRE could decrease the WNK4 expression, leading to a poor inactivation of the sodium-chloride channel (NCC) in renal collecting duct, which increases the renal sodium reabsorption. miRNA: Specific miRNAs can induce downregulation of target genes. For example miR-21, miR-24, and miR135a have been associated with genes that impair pathways associated to blood pressure control.

suppresses gene expression, whereas 5hmCyt generally correlates with gene activation [45]. So far, very little notion is available for the function of hydroxymethylation in human AH, although, considering its role as an intermediate product of DNA methylation and demethylation pathway and gene expression regulation, it is likely that future studies in this field will help in deepening the understanding of the role of DNA hydroxymethylation in AH possibly both as a pathogenetic and/or prognostic factor of disease.

Gene-Specific DNA Methylation in Hypertension DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Tremendous advances have been made in our understanding of the role of DNA methylation in development [62], transgeneration propagation of epigenetic profiles [63], cancer pathogenesis [64,65], and a number of other research areas, such as chronic diseases. AH has been raised as a disease associated to epigenetic regulation, where also genetic

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and nutritional factors can impair the proper control of blood pressure (BP). Here, several genes have been associated to epigenetic modifications in AH such as DNA methylation (Table 7.1), histone modifications (Table 7.2), and noncoding RNA/microRNA (miRNA) activity (Table 7.3). It has been well established that DNA methylation at gene-specific sites influences gene expression by directly interfering with transcriptional factor binding complexes or by histone modifications mediated by methyl-CpG binding proteins [22,89]. Increased importance has been devoted to mineralocorticoids and glucocorticoids in the onset and progression of AH [15], including studies associated to gene expression regulation of either aldosterone or cortisol synthesis, metabolism and effect over of mineralocorticoid receptor (MR) and glucocorticoid receptor (GR) [90]. A special interest has been dedicated to the pre-receptor metabolism performed by the 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD2) whose function is that of catalyzing the dehydrogenation of 11β-hydroxyglucocorticoids to 11keto-steroids by using NAD1 as a cofactor [90
93]. A partial or total deficiency of 11β-HSD2 activity may lead to the occupancy of MR by cortisol, causing sodium reabsorption in renal epithelium [94,95] and MR activation in non-epithelial cells (i.e., SMC, endothelium) [96], affecting adequate BP control [94]. An impaired MR stimulation by cortisol is the pathogenetic mechanism of a rare form of SS hypertension caused by mutations within the HSD11B2 gene that leads to a considerable or even a complete loss of the enzyme activity [95,97
99]. Deleterious homozygous mutations in the HSD11B2 gene are known to be associated with an apparent mineralocorticoid excess syndrome [100] and, when at the heterozygous state they could explain, although only partially, the impaired enzyme function [101]. The utilization of the intrauterine growth restriction (IUGR) rat model of hypertension, a condition that is known to increase the risk of adult hypertension development, helped to demonstrate an increased promoter methylation at HSD11B2 site [46]. In human placentas, the methylation levels at HSD11B2 promoter were considerably higher in the IUGR newborn rodents than in controls and related to a lower HSD11B2 expression, as expected by the usual repressive effect of hypermethylation at gene promoter sites [47]. The methylation setting at different CpGs correlated also with fetal growth parameters such as weight at birth and ponderal index, showing therefore that the promoter methylation at HSD11B2 site may be linked to the development of IUGR and it can be potentially associated to a higher risk of future development of hypertension [47]. Results from another human study evaluating the epigenetic regulation in HSD11B2 gene showed that promoter methylation of HSD11B2 is related to hypertension [25]. Methylation at HSD11B2 promoter measured in DNA extracted from PBMCs of hypertensive patients is inversely related to the enzyme function, showing an indirect proof for the role of HSD11B2 promoter hypermethylation in gene repression [25].

TABLE 7.2 Main Histone Modifications in AH Epigenetic Phenomenon

Gene

NCBI ID

MIM

Epi-Mark

Status

Sample Source

Affected Function Related to AH

Reference

Histone modifications

HSD11B2

NG_016549

614232

H3K6 methylation

Low

Rat, IUGR

Renal sodium balance

[46]

Alpha-ENaC (SCNN1A)

NG_011945

600228

H3K79 methylation

High (DOT1)

Murine

Renal sodium balance

[66
68]

LSD1 (KDM1A)

NM_001009999

609132

H3K4 methylation

Low

Mice, high salt

Enhanced vascular contraction

[69]

WNK4

NG_016227

601844

H3K

Low (NCC)

Murine

Poor inactivation of NCC (TCD). Increase sodium reabsorption

[70
73]

High

Rat (Wistar)

Vascular tone regulation

[74]

High

SHR

RAAS

[75]

SHR

Ionic transport (ubiquitous)

[76]

H4K Endothelial nitric oxide (NOS3) ACE1

163729

H3K27 3-methylation

NC_005118

106180

H3 Acetylation

NKCC1 (SLC12A2)

600840

H3K4me3

High

H3

High

Acetylation H3 3-methylation

Low

TABLE 7.3 Schematic Overview of the Main miRNAs in Relation to AH Target Gene

Sample Source

Affected Function Related to AH

Reference

miR-21

AGT

H295R cells

Aberrant regulation of Ag-II

[77]

miR-21

JAG1

HMVEC-D

Intracellular signaling

[78]

miR-92a-3p, miR-92b-3p, miR-99-5p, miR-100-5p

NOX 4

miR-124/135

MR (NR3C2)

Rat

Renal sodium balance

[79]

miR-24/34a

UCP2

SHR

Renal damage

[80]

miR-320/26b

IGF1R

Dahl SS rat

Increased fibrosis

[81]

miR-320/26b

PTEN

Dahl SS rat

Increased fibrosis

[81]

miR-181

REN

BPH/2J mice cells

Sympathetic activation

[82]

miR-132/212

AT1R (AGTR1)

Rats

AGT1R downregulation

[83]

miR-24

CYP11B1

Human adrenal

Cortisol synthesis

[84]

miR-24

CYP11B2

Human adrenal

Aldosterone synthesis

[84]

miR-23/34

TASK-2

Human adrenal

Modulation potassium homeostasis in APA cells

[85]

miR-30

Dll4

Zebra fish model

Vessels resistance

[86,87]

miR-29a39

MMP-2

Dahl SS rat

Renal fibrosis

[88]

Vessel resistance

BPH/2J, blood pressure hypertensive mice; HMVEC-D, human dermal microvascular endothelial cells.

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By considering the well-established role of ion flux mechanisms alterations in the pathogenesis of hypertension, Lee et al. analyzed promoter methylation of the Na1/K1/2Cl2 co-transporter 1 (NKCC1, official gene symbol Slc12a2; MIM 600840), a gene encoding for a solute carrier that transports sodium, potassium, and chloride through the cellular membrane [48,49]. The methylation status of NKCC1 promoter was measured in the aortas and hearts of spontaneously hypertensive rats (SHRs) by comparing them to Wistar Kyoto rats that were used as controls [48]. The authors confirmed that NKCC1 expression is upregulated by a mechanism induced by gene promoter hypomethylation in a spontaneously hypertensive rodent model [48]. Afterwards, Cho et al. [50] in 2011 showed that the regulation of NKCC1 was upregulated during postnatal development of hypertension by an epigenetic function. These results encourage studies in deepening the understanding of methylation status at NKCC1 gene in hypertensive patients. Riviere et al. [51] recently analyzed the somatic angiotensin-converting enzyme (sACE) gene expression regulation by promoter methylation by evaluating peripheral blood DNA and cell cultured models (Table 7.1). The sACE gene is a key regulator of BP by catalyzing the conversion of angiotensin I into physiologically active angiotensin II (Ang II), a substance with potent vasopressive properties. The higher levels of promoter methylation levels of sACE in cultured human endothelial cell lines and in rats in vivo were associated with transcriptional repression, therefore highlighting a possible involvement of epigenetic mechanisms through sACE methylation in hypertension [51]. In a human cell culture model using adrenocortical cells, the DNA methylation status was shown to be diminished by stimulation with interleukin 6 at a CCAAT/enhancer binding protein (CEBP) binding site and a transcription start site causing the expression activation of the angiotensinogen (AGT) gene [52]. AGT is the primary substrate of the RAAS and is, therefore, one of the major target proteins for the study of hypertension. The demethylation was accompanied by increased chromatin accessibility of the AGT promoter therefore switching the AGT phenotypic expression from an inactive to an active state. In a rat model, the high salt intake induced a decreased DNA methylation activity in rat visceral adipose tissue. Furthermore, an elevated circulating aldosterone concentration upregulates AGT expression and was associated with DNA hypomethylation around a CEBP binding site and a transcription start site also in human visceral adipose tissue, therefore showing that excess circulating aldosterone concentrations as well as a high salt intake act as a nutritional stimulus for the AGT gene expression in adipose tissue both in rats and humans [52]. Aldosterone is a mineralocorticoid hormone that act through MR altering the Na1 transport in collecting ducts of the kidneys, thus altering water uptake, blood volume, and eventually BP regulation. Excess of aldosterone contributes to AH, congestive heart failure, chronic kidney disease, coronary

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artery disease, and stroke [102
104]. Beside the already known genomic and nongenomic actions of aldosterone in renal and vascular cells [105
107], it has recently been shown, in cell culture models, that aldosterone could act also through an epigenetic way that alters the expression of epithelial Na1 channel subunit α (ENaCα) by modulating the DOT1L/AF9 repressive complex through epigenetic phenomena [66,67,104]. Promoter methylation of α-adducin gene (ADD1) was evaluated for the risk of developing AH [53]. A lower DNA methylation at ADD1 promoter site was found to be related to an increased essential hypertension risk while a differential power in predicting the risk of hypertension was observed for different sites of specific CpG sites of methylation in males compared to females [53]. Chronic hypoxia is related to modulation of estrogen receptor alpha (ESR1) gene methylation and risk of pre-eclampsia, where hypertension plays a major pathogenetic role, in an animal model [54]. The findings of an involvement of DNA methylation at several gene promoter sites in AH studies mainly derive from cell culture and animal model studies. This is to be expected since one of the main limitations for an approach in humans is due to the tissue specificity of epigenetic mechanisms that would require an invasive approach. Recent studies in humans are in fact evaluating profiles of DNA methylation by analyzing methylation signatures by a more feasible approach as the study of PBMCs-derived DNA. A promising approach seems also that of evaluating methylation marks from DNA obtained from biological fluids. Whether those methylation profiles relate to AH pathogenesis or may be useful for diagnostic and prognostic approaches certainly need further investigation.

Histone Modifications and AH Until not long time ago, the meaning of histone proteins was only thought as being functional to DNA packaging into the cell nucleus while a much more fascinating role for nucleosome unit forming proteins has being recently highlighted [108]. Posttranslational modifications such as acetylation, methylation, phosphorylation, ubiquitination at histone tail sites are key epigenetic phenomena and have been also described in cardiovascular diseases including AH (Fig. 7.1) [25,26,38,41,46,69]. All of those posttranslational histone modifications are features that occur epigenetically and contribute to regulate gene expression through the modification of chromatin structure (Fig. 7.1) [29,109,110]. Their contribution to gene function and regulation in AH is mostly to be explored, even though some studies recently showed their possible role in this disease [111]. A summary of the main epigenetic histone modifications documented in AH is shown in Table 7.2. A recent example, in AH associated with salt sensitivity is the with-no-lysine kinase 4 (WNK4), a serine-threonine kinase, which is a negative regulator of the thiazide-sensitive sodium chloride

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cotransporter (NCC). Under normal conditions, WNK4 inhibits NCC activity and leads to a decrease in sodium reabsorption in the distal convoluted tubule segments to maintain normal BP [70]. The GR plays a key role in β-adrenergic receptor (B-AR) stimulation-induced WNK4 downregulation and SS hypertension [112,113]. Epigenetic modulation is involved in the of BAR
GR
WNK4 pathway, which according to murine studies, is associated with activation of histone deacetylase 8, which decreases the binding of the acetylated histones 3 and 4 to the promoter of the WNK4 gene, which decreases the transcriptional activity of WNK4 through the recruitment of GRs to a negative glucocorticoid response element (nGRE) [71
73,114] In a rodent model, it has been reported that mice fed a high-salt diet, lysine-specific demethylase-1 (LSD1) deficiency was associated with AH [69]. LSD1 induces demethylation of histone H3 lysine 4 (H3K4) or H3K9 and thereby alters gene transcription. During the high-salt diet, LSD1 deficiency is accompanied by hypertension, enhanced vascular contraction, and reduced relaxation via nitric oxide
cGMP pathway, therefore supporting a function of LSD1-mediated histone demethylation in altering the NOS/guanylate cyclase gene expression and BP while mice are fed a high-salt diet [69]. In a IUGR rat model, that is an at-risk form for hypertensive disease in adulthood, besides modifications of methylation at HSD11B2 promoter site [46], a decreased trimethylation of H3K36 in exon 5 of HSD11B2 associated with decreased transcriptional activity has been also observed, then allowing to speculate that histone modifications may play a major role in the in utero reprogramming and a possible underlying mechanisms for hypertension in adult life [46]. The expression of ENaCα has been demonstrated to be epigenetically regulated by modulating the DOT1L/AF9 repressive complex. The methyltransferase DOT1 hypermethylates the lysine residue 79 on histone 3 (H3K79), which in presence of AF9, a DNA-binding protein, confers selectivity and represses the expression of a subset of genes for nucleosomes. One such gene encodes for a sodium channel subunit, the ENaCα. Aldosterone induces also the expression of serum- and glucocorticoid-induced kinase 1 (Sgk1), which phosphorylates AF9 and disrupts the complex. These steps are necessary and sufficient to increase, as shown in cell culture models, the ENaCα expression in the presence of the hormone
receptor complex [66,67,104]. H3K79 methylation contributes together with genetic variations to BP response to hydrochlorotiazide in humans showing a significant association between DOT1-like histone H3K79 methyltransferase (DOT1L) and an abnormal response to hydrochlorothiazide in AH [68]. In the renal artery of a SS hypertension model of the Wistar rat, it has been shown an upregulated expression of H3K27me3 with a possible functional effect in improving the hypertensive status of the rodent model [115]. The histone acetylation and methylation was also described as a regulatory mechanism for endothelial nitric oxide synthase (eNOS), an enzyme that is primarily responsible for the regulation of vascular tone and very much

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highlighted for its function in arterial hypertensive disease [74]. The study showed that the eNOS core promoter of endothelial cells was enriched in lysine 9 of histone H3 and lysine 12 acetylation of histone H4, and of di- and tri-methylation of lysine 4 of histone H3. The authors observed that those epigenetic histone modifications were significantly important for the expression of eNOS gene [74]. Moreover, Lee et al. [75] demonstrated that the Ace1 promoter regions of the spontaneously hypertensive rats tissues showed higher H3Ac and H3K4me3 possibly linking the tissue-specific Ace1 upregulation to histone modifications. The Nkcc1 gene was also described to be upregulated by an epigenetic mechanism involving histone modification as shown in the aortas of Sprague Dawley rats where the levels of the membrane transporter Nkcc1 mRNA and protein were significantly increased after administration of an Ang II infusion and found that the acetylated histone H3 was significantly increased in parallel with a decreased trimethylated histone H3. These observations suggest that histone modifications have a role in the epigenetic upregulation of Nkcc1 for the development of AH [76]. It is to be highlighted that the diagnostic and prognostic potential of epigenetic histone marks in human AH shows several challenges due to the low stability of histone modifications and to potential technical difficulties related to the use of antibodies to test histone marks which may vary significantly in terms of performance and lead toward reduced reproducibility of results, especially in human studies.

Noncoding RNAs and AH Regulatory, noncoding RNAs may give an important contribution to epigenetic regulation of gene expression (Fig. 7.1) [28]. Noncoding RNAs and particularly long noncoding RNAs may play an essential role in conferring site- or gene specificity to epigenetic modifications [40,111,116,117]. The role of noncoding RNAs has been recently described as having a function in cardiovascular diseases, including AH, even as possible marker of disease risk and progression [27,40,111,118]. Little is known thus far on the function of noncoding RNAs among which are enclosed the small noncoding RNA molecules, namely, miRNAs [111]. Their function is that of posttranscriptional regulators that work as guide molecules, pairing with partially or fully complementary motifs in 30 untranslated regions (UTRs) of their target mRNAs, leading to inhibition of translation or mRNA degradation. Table 7.3 shows the main epigenetic modification related to noncoding RNAs in AH. One of the first reports on the possible link of miRNAs function in AH comes from a work by Romero et al. [77] who studied, using a cell culture model of human adrenocortical cells, the link of Ang II (AGT) with miRNAs in steroidogenesis and proliferation associated to aldosterone secretion, known to be a main pathway for hormonal regulation in hypertensive

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disease. In that study, the authors observed that miR-21 expression is upregulated by AGT, and its overexpression caused an increase in aldosterone secretion and cell proliferation [77]. They speculated about alterations in miR-21 expression levels or function potentially involved in aberrant regulation of AGT signaling and abnormal aldosterone secretion by adrenal glands in humans leading to primary aldosteronism and hypertension [77]. In a recent study planned to identify endogenous miRNAs in endothelial cells that regulate mRNAs encoded by genes relevant to hypertension, the transfection of endothelial cells with anti-miR-21 oligonucleotides induced a significant suppression of the abundance of mRNA encoded by JAG1 (a gene involved in intercellular signaling) [78]. Interestingly, treatment with anti-miR-21 decreased BP in mice fed a 4% NaCl diet [78]. Still miR-21 was evaluated in three small case-control studies in patients with hypertension, and expression levels of miR-21 were significantly upregulated in hypertensive patients compared with normotensive subjects [119
121]. miR-21 level was positively correlated with the clinical systolic BP [119,121], clinical and 24 h diastolic BP [121], dipping status at the ambulatory BP monitoring [119], inflammation markers (C-reactive protein), and subclinical atherosclerosis [120]. miR-21 was also negatively correlated with markers of integrity of endothelial vasodilatation (NO and eNOS) [119
121]. A recent study demonstrated that miR-124 and miR-135a suppress the 30 UTR reporter construct activity of the MR NR3C2, a gene involved in familial hypertension and renal salt-balance maintenance [79]. Under a highsalt diet, in a rodent model using the stroke-prone spontaneously hypertensive rat (SHRsp) and stroke-resistant SHR strains, a differential modulation of renal uncoupling protein 2 (UCP2) gene and protein expression levels was observed along with rattus norvegicus (rno)-miR-24 and miR-34a gene expression, therefore highlighting a possible role through epigenetic regulation of UCP2 gene, a possible target for prevention of renal damage in a context of hypertensive disease in a rodent model [80]. The increase of miR-320 and miR-26b and decrease of miR21 have been observed in a salt-induced AH rodent model (Dahl SS rats), by analyzing aortas using a microarray technique. Interestingly, the use of β1-selective blocker (nebivolol) and selective β3-adrenoceptor antagonist S(2)-cyanopindolol determined a reversion of the phenomenon while atenolol (a pure β1-blocker) combined with specific β3 agonist BRL37344 restored the expression of all three miRNAs, similarly to the effect of nebivolol [81]. The insulin growth factor-1 receptor (IGF1R) was described as a putative target of miR-320, and phosphatase and tensin homolog on chromosome ten (PTEN) as a putative target of miR-26b and miR-21 where a highsalt diet caused vascular remodeling and increased fibrosis possibly due to downregulation of IGF1R and upregulation of PTEN in the rat aortas [81]. In a different model of genetically hypertensive mice (BPH/2J) where the hypertension is caused by an overactivation of the sympathetic nervous

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system, a 1.6-fold higher renal renin mRNA was found to be associated with a 0.8-fold lower abundance of miR-181a, identified previously as regulating human renin mRNA, therefore proposing the hypothesis that enhanced sympathetically induced renin synthesis may be mediated by lower miR-181a therefore significantly contributing to the inset of hypertension in BPH/2J mice [82]. The importance of miR-181a as a potential novel mediator in hypertension was recognized by the results of a previous human study by Marques et al. [122] who showed, by analyzing tumor-free samples of kidney tissue obtained from patients affected by renal cancer, that hypertensive subjects markedly underexpressed the miR-181a together with a significant overexpression of renin mRNA. In human endothelial cells, endogenous miR-181a was also responsible of a significant suppression of the abundance of mRNA encoded by ADM [78]. ADM is the gene encoding the adrenomedullin peptide, initially isolated from the adrenal gland, but produced also in the vasculature, with numerous actions, including vasodilation, natriuresis, anti-apoptosis, and stimulation of NO production. This finding could be relevant in the comprehension of miRNAs modulation of genes related to hypertension considering that adrenomedullin plasma level was previously demonstrated elevated in patients with hypertension and heart failure [123,124]. Knowing the major role played by the corticosteroid hormones such as cortisol and aldosterone in the regulation of BP, a recent study evaluated the regulation by epigenetic mechanisms via miRNA of two genes encoding for enzymes that are strictly involved in the controlled release of corticosteroids from the adrenal cortex: the 11β-hydroxylase (CYP11B1) that is responsible for the terminal conversion that produces cortisol and the aldosterone synthase (CYP11B2) that fulfills the aldosterone production. Knocking down the Dicer1, a key enzyme in miRNA maturation by siRNA technique, CYP11B1 and CYP11B2 expression appeared significantly modified [84]. Several putative binding sites for several miRNAs were observed, including miR-24, in the 30 UTR of CYP11B1 and CYP11B2 mRNAs. In vitro manipulation of miR-24 confirmed its ability not just to modulate CYP11B1 and CYP11B2 expression, but also the production of cortisol and aldosterone [84]. The miRNA expression has been also associated to aldosterone secretion. Recently Lenzini et al. conducted an elegant study, which analyzed the transcriptome and the miRNA profiles of 32 consecutive hypertensives patients with primary aldosteronism due to aldosterone-producing adenoma (APA), and investigated the protein expression and localization of TWIK-related acid-sensitive K1 channel 2 (TASK-2) in APA and adrenocortical cell lines [85]. Primary aldosteronism is the most common cause of secondary hypertension, the exact molecular mechanisms of the disease are unknown, but alterations on potassium channels, such as TASK-2, may have an important role [125]. TASK-2 was less expressed at the transcript and protein levels in APAs than in the normal human adrenal cortex, and H295R cell transfection with a TASK-2 dominant-negative mutant construct was able to increase the

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aldosterone production by 153% and the gene expression of aldosterone synthase. At the same time, it was demonstrated that two miRNAs, hsa-miR23 and hsa-miR-34, studied by a luciferase assay, decreased the TASK-2 expression by binding to the 30 UTR of the TASK-2 gene. The authors, considering that the luciferase signal was lowered by 35% and 22% with hsa-miR23 and has-miR-34a, respectively, but not abolished, suggested a high degree of complexity of this regulation, with different miRNAs probably playing additive effects in modulating TASK-2 expression in the APA cells [85]. In a study using a zebra fish model, Jiang et al. [86] investigated the function of miR-30 family members in arteriolar branching morphogenesis via delta-like 4 (Dll4)-Notch signaling. The miR-30 family that consists of five miRNAs members that are highly conserved between humans and zebra fish with complete base-pair matching with the 30 UTR of Dll4 mRNA in these species seems to interfere with BP control by the regulation of the endothelial tip cells by directing them either toward pro-angiogenic or antiangiogenic processes, with evidence that the rarefaction of small arterioles and capillaries relates to increased peripheral vessels resistance and is detected in early stages of hypertensive disease relating, therefore, the regulation of blood vessels growth to hypertension [86]. Although the exact significance of these findings in human hypertension needs further investigation, it is indeed interesting to point out that treatment with anti-angiogenic drugs leads toward higher arterial BP and the control by miR-30 may be one of the possible pathways involved [87]. An influence on vascular resistance was also hypothesized by the miRNAs modulation of endothelial-derived oxygen reactive species. Endogenous miR92a-3p, miR-92b-3p, miR-99-5p, and miR-100-5p were found to reduce NOX4 mRNA abundance in endothelial cells and reduced H2O2 release [78]. Among the major pathways regulating BP, certainly a major role is played by the renin
angiotensin
aldosterone hormonal pathway where Ang II controls BP and fluid homeostasis through stimulation of aldosterone by the function of two main regulatory receptors, AT1R and AT2R where the classical Ang II responses for BP control are mainly mediated by the AT1R. Tissues with an impact on BP control, including vessels and kidneys, express Ang II receptors. In a study in which heart, aortic wall, and kidney tissue of female Sprague Dawley rats with hypertension were analyzed, miR-132 and miR-212 expression were highly increased in the left ventricle, aorta, and kidney, as well as in the plasma in Ang II-induced hypertension in rats [83]. However, a decreased expression of miR-132 and miR-212 was observed in human internal mammary arteries obtained from patients who underwent cardiac arteries bypass grafting and treated with AT1R blockers, whereas differently from the rodent model, no effect was seen in those treated with β-blockers. More experiments are warranted to explain those results and in particular whether there may be a different functional role of those miRNAs in humans compared to the animal model in relationship to the regulatory effect of those specific

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miRNA in gene expression regulation potentially involving differential transcriptional system in humans versus the rat model [83]. Considering the kidney hypertension-related target organ damage, documented by renal fibrosis and microalbuminuria, there is some evidence of a potential molecular regulation by miRNA [88,126]. In a well-known experimental model of Ang II-dependent hypertension (Sprague Dawley rats), it was studied the role of the Ang II-induced differential miRNA expression in renal glomerular and tubulo-interstitial fibrosis. As expected Ang II treatment increased systolic BP (p , 0.0001), albuminuria (p , 0.01), and both glomerular and tubulo-interstitial fibrosis. At the same time, at microdissection microarray analysis, miR-29a-3p was downregulated in renal tubules and this result was confirmed by real-time polymerase chain reaction experiments. Matrix metalloproteinase-2 (MMP-2) was identified as putative miR-29a-3p target and functionally confirmed by luciferase activity assay, suggesting that the development of renal fibrosis depends on different molecular mechanisms that could include miRNA modulation [88]. In peripheral blood cells isolated from untreated patients with recently diagnosed essential hypertension, it was demonstrated that the gene expression level of miR-208b and miR-133a was strongly correlated with urinary albumin excretion levels. The authors highlighted the perspectives on the development of a new generation of biomarkers for the monitoring of endorgan damage in hypertension [126]. In Table 7.3 are summarized findings on miRNAs with a hypothesized target gene and altered function in relation to hypertension. Considering the complexity of metabolic pathways involved in hypertension development, several and yet unknown different noncoding RNAs may certainly be involved in gene regulatory processes related to AH. An issue, in this regard that should be taken into account to push forward the research in this field, is that miRNAs may serve as potentially useful biomarkers of AH disease development and progression with possible significant clinical implications. Given the discovery that miRNAs are not only involved in the regulation of cellular processes in the miRNA-producing cell, but that they also regulate processes in other cells by exosomal transfer [127], the diagnostic and therapeutic applications of miRNAs have gained increasing attention. Intracellular levels of miRNAs have been extensively mapped in a number of diseases. Among the most studied diseases in this regard is cancer, and lately cardiovascular diseases [128], in which specific miRNAs are often overexpressed or underexpressed compared with healthy cells. Identification of miRNA mainly in exosomes of different biofluids, highlights the teragnostic potential of miRNA signatures to identify early epigenetic marks or signs of the hypertensive disease progression [129]. Exosomes and microvesicles are involved in a large variety of body processes [130]. They are concentrated carriers of genetic and proteomic information and thus are believed to play important roles in intercellular communication, with high diagnostic and therapeutic potential [131].

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CONCLUSIONS A growing evidence shows that epigenetic phenomena are mechanisms possibly underlying, at a molecular level, AH disease development. There is, however, a real need for deepening our understanding in this field. Studies on epigenetic features of DNA for their link to hypertension rely mostly on cell culture and animal models, so far, thus more numerous and larger human studies are especially necessary to investigate the influence of epigenetic mechanisms in AH-related gene regulation to identify molecular pathways of significance for the better understanding of hypertensive disease development and progression. An important issue for future research pertains also to define epigenetic profiles as useful biomarkers in humans from adequate target tissues, as well as easily available, such as PBMCs. Since epigenetic signals are potentially modifiable by environmental/nutritional factors there is ample expectation for innovative preventive and/or therapeutic policies.

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