*3.1. Epigenomic Regulation in the Whole Vessel*

Several epigenomic studies have been conducted at the level of the whole vessel in animals. The Na+–K+–2Cl<sup>−</sup> cotransporter 1 (NKCC1), which is encoded by *Slc12a2*, regulates the exchange of sodium, potassium, and chlorine ions across cells of various types, including VSMCs and endothelial cells, regulating ionic balance and cell volume [64]. It has been showed that NKCC1−/<sup>−</sup> mice displayed a >15 mmHg reduction in systolic BP compared to wild-type and reduced ability to maintain vascular tone [65]. Pharmacological inhibition of NKCC1 by bumetanide led to an immediate 5% reduction in BP, highlighting its importance in BP regulation [66]. Combined bisulfite restriction assay and bisulfite sequencing revealed that the *Slc12a2* promoter was hypomethylated in SHR aorta and heart compared to WKY, which resulted in an upregulation of NKCC1 in both mRNA and protein levels detected by PCR and western blots in SHR vs WKY [11].

Furthermore, DNA methylation has been also linked to the estrogen receptor-mediated vascular regulation. Estrogen induces vasodilation as well as inhibits the response of blood vessels to injury, by interacting directly with the vasculature [67]. Two estrogen receptors have been identified: estrogen receptor α (ERα) and estrogen receptor β (ERβ). In uterine arteries of pregnant sheep, hypermethylation of the ERα (*ESR1*) promoter during hypoxia reduced its expression, leading to preeclampsia and impaired cardiovascular homeostasis [12]. Promoter hypermethylation inhibited transcription factor binding and promoter activity, leading to gene repression [12]. Although these mechanisms need to be confirmed in the human, this evidence suggests that the protective nature of estrogen signaling may be regulated through epigenetic mechanisms, which may also contribute to the gender disparity in hypertensive patients.

Moreover, histone modification has been found to be involved in the regulation of vascular function in hypertension. Additionally, NKCC1 can also be regulated by histone modification. Angiotensin II (Ang II) was delivered in rats, in vivo, to increase BP and the changes in NKCC1 mRNA, protein and epigenetic modifications at the *slc12a2* promoter were measured in the aorta [23]. Real-time PCR and western blot revealed a progressive increase in NKCC1 expression over the period of Ang II delivery. Interestingly, H3Ac levels were consistently increased in Ang II infused rats whereas H3K27me3 (a repressive histone code) levels were decreased as compared to sham [23]. These results together suggest that NKCC1 might be regulated by both DNA methylation and histone modification [4].

Overexpression of SIRT1, a histone deacetylase, reduced angiotensin-II induced hypertrophy, in vitro, and vascular remodeling and hypertension, in vivo [68,69]. Among the affected parameters were reduced reactive oxygen species (ROS) generation, vascular inflammation, and collagen synthesis in arterial walls. SIRT1 overexpression also decreased the association between nuclear factors on specific binding sites on TGF [69].

In addition to these studies on the whole vascular wall in hypertensive animal models, many observations on the epigenomic regulation have been done on specific cells of the vascular wall or the cells interacting with vessels such as the blood cells circulating around the body. We next outline these investigations.
