*3.2. miRNAs in Endothelial Dysfunction*

Vascular endothelial dysfunction is highly associated with hypertension. Endothelial cells function to release vasodilators into the blood stream to reduce vascular resistance. As these cells face continuous hemodynamic forces with shear stress and stretch, they play an important role in development, regulation and remodeling of the vasculature. Increased blood pressure can alter the phenotype and function of the endothelial cells [82]. With endothelial dysfunction, there is a reduced vasodilatation, activation and release of inflammatory factors, an increase of reactive oxygen species (ROS), a reduction of nitric oxide, which then develops into increased vascular tone, increased vascular stiffness and pulse pressure, and sustained elevated blood pressure [83]. The maintenance of the endothelial cell phenotype involves a number of molecular players such as protein kinases, integrins, endothelial nitric oxidase synthase (eNOS) and NO, vascular endothelial growth factor (VEGF) and miRNAs [84,85]. Evidence has emerged that miRNAs are involved in angiogenesis, the proliferation and function of endothelial cells and their dysfunction.

Several human studies have shown the association of miRNAs and hypertension in the aspect of endothelial dysfunction. In experimental models, change in expression of an L-arginine transporter gene, *SLC7A1*, alters NO production and induces endothelial dysfunction. An SNP of a novel C/T polymorphism in the 3 UTR of *SLC7A1* is found in 278 essential hypertensive subjects (T allele frequency: 13.3%), compared with the 498 normotensive subjects (T allele frequency: 7.6%) [51]. The impact of the T allele is such that it disrupts the binding of transcription factor SP1, extends the 3 UTR length to increase the binding site of miR-122, which in turn reduces *SLC7A1* expression. A tumor-suppressive miRNA, miR-505, was found to be increased in hypertensive patients from three independent cohorts [52]. The increase of miR-505 represses fibroblast growth factor 18 (FGF18), a proangiogenic factor in the endothelial cells that promotes endothelial migration and thus disables the migration and tube formation of endothelial cells [53]. Another study shows that vascular endothelial cells release circulating miRNAs to combat pathogenic virus. From plasma collection of a hypertensive and normotensive Chinese population, three significant miRNAs were isolated: human cytomegalovirus (HCMV) miRNA (hcmv-miR-UL112), miR-296-5p and let-7e. Based on in vitro transfection of HEK293 cells with the reporter gene constructs, the interferon regulatory factor 1 has been reported to be the target of hcmv-miR-UL112 [54]. Interestingly, hypertensive patients showed increased HCMV seropositivity and quantitative titers (52.7% vs. 30%, *p* = 0.0005; 1870 vs. 54 copies per 1 mL plasma, *p* < 0.0001), which suggest that cytomegalovirus causes the release of hcmv-miR-UL112 from vascular endothelial cells. The group suggested a possible new link between HCMV infection and essential hypertension [54].

There is some evidence of the involvement of miRNA and eNOS production in hypertension and in endothelial dysfunction. eNOS is responsible for the production of NO within the endothelium. Inhibition of eNOS decreases NO availability and increases oxidative stress, endothelium dysfunction and hypertension. A study reported that inflammatory release of tumor necrosis factor-α (TNF-α) induces the transcription of miR-155, which targets the 3 UTR of eNOS to inhibit its expression. Inhibition of miR-155 reverses the effect of eNOS downregulation [55]. Furthermore, the miR-221/222 cluster, known as the sensitive regulator in the endothelium, regulates the NO release by its binding to the 3 UTR of eNOS mRNA in endothelial cells, as well as other genes, such as activator STAT5a, transcription factors Ets1, Es2 and cyclin-dependent kinase cell cycle regulators p21Cip1 and p27Kip1 [56–58]. Recently, it was found that the presence of pro-inflammatory cytokines upregulates miR-146a and miR-146b. Their upregulation not only inhibits endothelial activation and represses NF-κB and MAP kinase pathways, but also targets HuR, which is an RNA binding protein that suppresses eNOS [59].

Interestingly, Dicer, the RNaseIII enzyme, not only plays a role in processing the premiRNA sequence into a shorter double-stranded miRNA, but also contributes to angiogenesis. The absence of Dicer in both the in vitro and in vivo system causes dysregulation in angiogenesis. There is a number of miRNAs that are also involved in angiogenesis. One of them is the endothelial-specific miR-126. The role of miR-126 includes: influencing the integrity and regulation of the growth of the vasculature by controlling the endothelial response to VEGF, inhibiting the negative regulators of the VEGF pathway and controlling the level of the vascular cell adhesion molecule (VCAM-1) for inflammatory adhesion [60–62].
