**4. Clinical Application of Epigenomic Studies in Human Systemic Hypertension**

Although VSMCs and ECs are the main components of the vascular wall, inflammatory cells and circulating blood cells also play essential roles in the development of hypertension, and their epigenetic regulation should be reported. As human studies increase and biomedical technology advances, many researchers have opted to use peripheral blood to isolate DNA and measure epigenetic programming. While one can speculate that there may be cell to cell or tissue to tissue variation within the human, these studies offer a powerful tool in exploring differences in epigenetic patterns from patient to patient and identifying potential risk factors and key marks. One of the first questions that researchers asked was whether there was a difference in the DNA methylation of hypertensive patients from normotensive individuals that can be detected in blood cells. Interestingly, the total 5mC level was shown lower in the peripheral blood cells of patients with essential hypertension, and even lower in patients with Stage 1 hypertension [85]. This finding indicates that some epigenomic signatures in hypertensive patients can be detected in the peripheral blood at an early stage of hypertension, thus providing a predictive biomarker for the development of hypertension. While initial DNA methylation studies focused on the global level of 5mC, subsequent research has shown that the epigenetic regulation of specific DNA sequences in peripheral blood cells can contribute to the hypertensive state.

Since hypertension is a multifactorial disease which may be mediated by alterations in multiple biological pathways, research continues to identify possible targets whose manipulation may have effects on systemic BP. It has been shown that DNA variation is correlated with DNA methylation. Genome-wide linkage and association studies (GWAS) have identified single nucleotide polymorphisms (SNPs), which influence BP. SNPs that create CpG sites may be targets for epigenetic modifications, just as loss of these sites will prevent DNA methylation. For example, a recent trans-ancestry genome-wide association study identified genetic variants at 12 new loci which have been differently DNA methylated in hypertension, four of these genes encoded proteins that participate in the regulation of vascular tone and VSMC plasticity, including: insulin like growth factor binding brotein 3 (IGFBP3), potassium two pore domain channel subfamily K member 3 (KCNK3), phosphodiesterase 3A (PDE3A), and PR domain-containing protein 6 (PRDM 6) [86]. It is remarkable that a two-fold enrichment between sentinel BP single nucleotide polymorphisms (SNPs) and DNA methylation was observed, highlighting the importance of genetic variation and providing an additional hypothesis for the racial disparity observed in the incidence of hypertension [86]. While additional studies are needed to confirm whether DNA methylation of these specific genes is enough to cause changes in BP, it is exciting to note that DNA methylation explains part of the relationship between sentinel SNPs and BP (*r* = 0.52; *p* = 0.005) [86].

In a study of the DNA methylation levels of 5 CpG dinucleotides in 62 patients, reduced promoter methylation of the α-adducin (*ADD1*) gene was been shown to be linked to an increased essential hypertension risk [14]. Females were shown to have higher methylation of the *ADD1* promoter, with a sharp decline in post-menopausal women, suggesting that estrogen signaling may also influence epigenetic regulation of the *ADD1* gene [14]. Although α-adducin is a cytoskeletal protein, an *ADD1* variant has been implicated in stimulation of renal sodium reabsorption and, subsequently, hypertension [87].

Epithelial sodium channel (ENaC), a nonvoltage-gated sodium channel, comprises three homologous subunits (α, β, and γ), with the alpha subunit (*SCNN1A*) being indispensable for channel activity [15]. The ENaC plays a critical role in maintaining extracellular fluid volume and BP. Hypermethylation of *SCNN1A* at CpG islands within the transcriptional region was associated with increased risk of developing hypertension. Importantly, the methylation level was regulated by age, gender, and antihypertensive therapy [15]. Hypermethylation of CpG islands within exons is typical of expressed genes, suggesting that *SCNN1A* gene is highly expressed in hypertension. Furthermore, CpG1 hypermethylation and CpG2 hypomethylation of the gene promoter of the amiloride-senstive sodium channel beta subunit (*SCNN1B*) were linked to hypertension, with the tested antihypertensive therapy only affecting CpG1 levels [16].

Toll-like receptors (TLRs) contribute to chronic inflammation, a mechanism that has be shown to play a role in the development of hypertension [17]. Hypomethylation of the *TLR2* promoter at CpG6 was shown to be associated with an increased risk of systemic hypertension [17]. Notably, an important link between environmental cues, such as alcohol consumption and smoking, and specific CpG islands were identified as the risk factors of hypertension [17]. The identification of methylation patterns as a result of behavior is significant since hypertension has often been described as a lifestyle disease but precise mechanisms by which chronic inflammation through TLRs contribute to hypertension are still unclear.

Euchromatic histone-lysine in methyltransferase 2 (EHMT2; also called G9a) is a lysine histone methytransferase found in leukocytes which regulates the expression of IL-17 through methylation pattern H3K9me2 [88,89]. In a longitudinal genome wide methylation study of Roux-en-Y bypass patients, the EHMT2 promoter was found to be hypomethylated in hypertension, suggesting that epigenetic regulation of EHMT2 contributes to vascular pathophysiology [18]. It remains to be explored whether IL-17 has effects in human hypertension and whether epigenetic regulation of EHMT2 is alter the hypertensive state.

Together, all of the evidence indicates that, although epigenetic regulations of systemic hypertension in the vasculature are far from fully understood, the study of epigenetic regulation in systemic hypertension holds valuable potential for the identification of biomarkers and may provide for useful therapeutic targets. The discovery of new drugs based on the epigenomic regulation of the hypertensive vasculature, both synthetic and natural, offers new hope to protect ourselves against the silent killer.

#### **5. Mechanisms Underlying Epigenetic Alterations in Hypertension**

It is well known that hypertension can be influenced by genetic and environmental factors. Although the mechanisms underlying the epigenetic alterations during the development of hypertension have not been completely elucidated, epigenetic programming caused by adverse fetal environments, in utero, has been shown to be strongly correlated with the hypertension in adult offspring [90]. For example, intrauterine exposure to a maternal low-protein diet in the rat results in the development of hypertension in adult offspring rats which is associated with hypomethylated *Atgr1β* gene promoters along with increased adrenal expression of AT1bR [7,91–93]. In addition, prenatal inflammation exposure due to the maternal inflammatory diseases is highly associated with adult hypertension in the offspring [94–97]. It has been also showed that prenatal exposure to lipopolysaccharide (LPS), a nonspecific immune-inflammatory stimulant, led to hypertension in adult offspring rats, correlating with the augmentation of histone H3 acetylation (H3AC) on the angiotensin-converting enzyme 1 (ACE1) promoter, which induced the upregulation of the (*ACE1*) expression in renal cortex tissues [98–100]. Importantly, prenatal anti-inflammatory treatment is able to prevent offspring from fetal programming hypertension [100]. These results together indicate that epigenetic alterations induced during the fetal development are crucial contributors to the development of hypertension.

In addition to these "congenital" effects, the epigenetic alterations can be acquired during the early period of life due to the increased vulnerability of the reprogramming cells to environmental stress, such as malnutrition, toxic chemicals, infections and mental stress. This concept is further supported by the epidemiological investigations on human population which showed that social and environmental stresses during the early period of life influence epigenetic processes that contribute to the adult race-based US health disparities in diseases including hypertension [101]. Furthermore, several lines of evidence suggest that environmentally-induced epigenomic alterations can be transmitted to subsequent generations with disease phenotypes [102]. These observations, together, indicate the interaction of the genetic and environmental factors on the epigenomic alterations in the development of hypertension. On the other hand, it has been shown that increased hemodynamic forces are able to induce DNA methylation and histone modification in VSMCs and ECs [103]. In accordance with these understandings, it is important to note that while the epigenomic alterations are a part of the mechanisms involved in blood pressure elevation, they can also be a consequence of blood pressure alterations. Additional studies are therefore needed to determine causative agents of human epigenomic manipulation and their roles in development of hypertension.

Despite this evidence, the mechanisms by which DNA demethylation or histone modification are involved in the gene specific regulation of hypertension have not been fully elucidated. One mechanism suggests that the cell specific DNA demethylation is linked to a unique response to hormone stimulation [104]. Several studies suggest that interactions with sequence-specific DNA binding proteins and co-repressor complexes can target certain proteins to histones in a gene-specific manner [105,106]. Furthermore, other studies have shown that in addition to histone methylases, there are multiple histone demethylases, such lysine-specific demethylase 1-(LSD1), which removes mono- or di-methyl groups from H3K4 and the Jumonji C-(JmjC) domain-containing demethylases 5, which removes the tri-methylated modification and demethylates histones in a gene-specific manner by interactions between demethylases and DNA sequence specific nuclear factor complexes. Moreover, recent studies have shown that specific histone demethylases may regulate androgen-mediated transcriptional responses [107–109]. However, further studies are needed to confirm these mechanisms in the hypertensive individuals.
