*3.2. Epigenomic Regulation in Vascular Smooth Muscle Cells (VSMCs)*

Vascular smooth muscle cells (VSMCs) play an integral role in the regulation of peripheral resistance by modulating vascular tone. Autonomic nervous activity as well humoral agents are able to regulate contraction of VSMCs, leading to changes in BP [70]. It has been noted that VSMCs play a key role in the pathophysiology of hypertension due to their remarkable ability to dedifferentiate, allowing them to switch between contractile and synthetic states, in response to environmental cues or stress [71]. Studies have shown that this phenotypic switch between contractile and synthetic states in VSMCs could be governed by epigenetic modifications [71,72]. In addition, our recent studies also showed the alteration of intrinsic VSMC stiffness contributes to the development of hypertension [68,69]. A thorough understanding of epigenetic regulatory mechanisms in the VSMCs is mandatory as we develop new antihypertensive therapies.

Ten-eleven translocation-2 (TET2), a key enzyme in the DNA demethylation pathway, is a major governor of SMC plasticity and is highly expressed in VSMCs [13]. TET2 knockdown leads to a decreased expression of contractile markers such as myocardin (MYOCD), serum response factor (SRF), and myosin heavy chain 11(MYH11) and an increase in proliferative markers such as kruppel like factor 4 (KLF4) and myosin heavy chain 10 (MYH10), while TET2 overexpression restored contractile markers and inhibited synthetic genes, suggesting that TET2 serves as an important switch in VSMCs [13]. Importantly, it was shown that TET2 binds to the promoters of MYOCD, SRF, and MYH11, implicating its role in demethylating contractile genes [13]. Stimulation of the TET proteins may offer therapeutic potential in hypertension and atherosclerosis where pathological VSMC switching have been described, and re-differentiation of VSMCs is needed.

In addition to DNA methylation, histone modifications are also found to be able to modulate gene expression in VSMCs. Serum response factor (SRF), a key mediator of SMC transcription, binds to highly conserved domains near gene promoters known as the SRF binding sites or CArG boxes. Recruitment of SRF to the CArG boxes of SMC marker genes has been implicated to be associated with hyperacetylation of H3 and H4 in SMC-differentiated cells [20]. SRF complexes with cAMP-response element-binding protein (CREB)-binding protein on the hyperacetylated *SM22* gene promoter, leading to its expression [73]. Moreover, p300 mediated-acetylation of myocardin is critical for its dissociation from the inhibitory effects of histone deacetylase 5 (HDAC5) [74,75]. Myocardin's acetylation enhances its binding of SRF and the CArG boxes and is required for VSMC gene transcription [76]. Interestingly, our recent studies showed that upregulation of SRF/myocardin in VSMCs mediates the intrinsic VSMC and aortic stiffness in hypertension [77,78], indicating the key role of this signaling pathway in the regulation of BP. The further investigation of the epigenetic regulation of SRF/myocardin signaling may be a promising target for future studies.

It is generally accepted that oxidative stress and mild chronic vascular inflammation contribute to the pathophysiology of hypertension [79]. Activation of the nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) inflammasome, a cytosolic complex for early inflammatory responses, generates proinflammatory cytokines such as interleukin 1 β (IL-1β) and interleukin 18 (IL-18) through the activation of caspase-1 [80]. These cytokines have been implicated in hypertension and consideration should be given to their therapeutic potential [81]. Recently, the *NLRP3* gene promoter was shown to have increased acetylation at lysine 9 of histone 3 and HAT expression in SHR VSMCs [21]. Importantly, inhibition of NFκβ and HAT by curcumin prevented the activation of the NLRP3 inflammasome, VSMC phenotypic switching, and proliferation in VSMCs of SHR [21]. These findings imply that inhibition of the inflammasome, IL-1β, IL-18, and HAT by reducing histone

acetylation at the *NLRP3* gene promoter may prove effective in controlling chronic inflammation in hypertension and reducing the pathology of the disease.
