**4. Endothelial cells**

Vascular endothelial cells (ECs) cover the internal surface of blood vessels. The primary role of ECs is the maintenance of vessel wall permeability and function as a sensor for the altered physical and chemical signals by physiological or pathological processes including thrombo‐ sis, inflammation, and vascular wall remodeling [80–82]. The knockdown of Dicer in human ECs resulted in the change of several key regulator proteins of EC biology and angiogenesis, such as TEK (Tie-2), KDR (VEGFR2), adhesion molecules and proteins, cytokine, chemokine, and IL-8. Furthermore, EC-specific knockdown of Dicer activated the endothelial nitric oxide synthase (eNOS) pathway, which reduces proliferation and capillary formation of EC, and the endogenous miRNA expression levels were also significantly altered [83]. These results indicate that altered miRNA expression in ECs affects the maintenance and functions of ECs (Figure 4).

**Figure 4.** miRNAs in ECs regulating during vascular remodeling.

#### **4.1. miR-126**

Adhesion molecules in ECs are directly associated with leukocyte trafficking to the region of the injury and/or inflammation [84]. Generally, resting ECs do not express adhesion molecules, but activated ECs express adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), which plays an important role in modulating leukocyte trafficking and inflamma‐ tion [85]. Based on microarray and Northern analysis, Harris et al. showed that miR-126 is the most frequently expressed in HUVECs as well as other origin ECs from dermal, brain, and vein [86]. *In silico* analysis suggests that VCAM-1 is one of the targets of miR-126. The upre‐ gulation of miR-126 by transfection of pre-miR-126 results in inhibiting TNF-α induced VCAM-1 expression, whereas the knockdown of miR-126 by antisense miR-126 oligonucleo‐ tide is causing TNF-α induced VCAM-1 upregulation in a dose-dependent manner. In addition, overexpression of miR-126 mediated a decrease of VCAM-1 downregulating leukocyte adherence to ECs. These data suggest that miR-126 regulates the expression of adhesion molecules and plays a crucial role in the control of vascular inflammation [86].

Fish et al. found that EC-specific miRNA miR-126 is also highly enriched in Flk-1 positive vascular progenitors from differentiating mouse embryonic stem cells in the embryonic body [87]. Nevertheless, overexpression of miR-126 using miR-126 mimic does not promote endothelial differentiation of embryonic stem cells. The loss-of-function of miR-126 by a morpholino antisense to miR-126 shows that the EC migration and the formation/stability of capillary tubes are decreased compared to normal HUVECs. In addition, EC-targeted deletion of miR-126 results in vascular abnormalities such as vascular leakage, hemorrhaging, and embryonic lethality in both zebrafish [87] and mice [88], because of reduced EC growth, sprouting, and adhesion by decrease in angiogenic growth factor signaling. The pro-angio‐ genic function of miR-126 was due to its ability to repress two negative regulators of angiogenic process, namely sprouty-related EVH1 domain-containing protein 1 (SPRED1) and phosphoi‐ nositol-3 kinase regulatory subunit 2 (PIK3R2) [87, 88]. Taken together, miR-126 is considered as new therapeutic implication for modulating vascular formation, function, leakage, and abnormal angiogenesis.

### **4.2. Endothelial Shear Stress (ESS) and miRNAs**

such as TEK (Tie-2), KDR (VEGFR2), adhesion molecules and proteins, cytokine, chemokine, and IL-8. Furthermore, EC-specific knockdown of Dicer activated the endothelial nitric oxide synthase (eNOS) pathway, which reduces proliferation and capillary formation of EC, and the endogenous miRNA expression levels were also significantly altered [83]. These results indicate that altered miRNA expression in ECs affects the maintenance and functions of ECs

Adhesion molecules in ECs are directly associated with leukocyte trafficking to the region of the injury and/or inflammation [84]. Generally, resting ECs do not express adhesion molecules, but activated ECs express adhesion molecules such as vascular cell adhesion molecule 1 (VCAM-1), which plays an important role in modulating leukocyte trafficking and inflamma‐ tion [85]. Based on microarray and Northern analysis, Harris et al. showed that miR-126 is the most frequently expressed in HUVECs as well as other origin ECs from dermal, brain, and vein [86]. *In silico* analysis suggests that VCAM-1 is one of the targets of miR-126. The upre‐ gulation of miR-126 by transfection of pre-miR-126 results in inhibiting TNF-α induced VCAM-1 expression, whereas the knockdown of miR-126 by antisense miR-126 oligonucleo‐ tide is causing TNF-α induced VCAM-1 upregulation in a dose-dependent manner. In addition, overexpression of miR-126 mediated a decrease of VCAM-1 downregulating leukocyte adherence to ECs. These data suggest that miR-126 regulates the expression of adhesion molecules and plays a crucial role in the control of vascular inflammation [86].

Fish et al. found that EC-specific miRNA miR-126 is also highly enriched in Flk-1 positive vascular progenitors from differentiating mouse embryonic stem cells in the embryonic body [87]. Nevertheless, overexpression of miR-126 using miR-126 mimic does not promote

(Figure 4).

64 Muscle Cell and Tissue

**4.1. miR-126**

**Figure 4.** miRNAs in ECs regulating during vascular remodeling.

The atherosclerotic lesions form at specific arterial regions such as the vicinity of branch points, the outer wall of bifurcations, and the inner wall of curvatures, where they show the disturbed flow and the change of hemodynamic forces, especially endothelial shear stress (ESS) [89]. ESS has been reported to regulate endothelial gene expression and promotes EC activation and atherosclerotic plaque progression and vascular remodeling [90, 91].

By miRNA microarray analysis using human umbilical vein endothelial cells (HUVECs), Ni et al. identified that the expression of miR-663 was mediated by low ESS (LSS) and LSS-induced miR-663 expression improved monocyte adhesion to ECs, but it had no effect on EC apoptosis. MicroRNA-663 has been reported to regulate several transcription factors related to inflam‐ matory responses, such as KLF4, C/EBPB, and ATF3 [92].

In addition, miR-21 was also upregulated by LSS at transcriptional level in HUVECs through an increased activity and binding of c-Jun, a component of transcription factor activator protein-1 (AP-1), to the promoter region of miR-21 [93]. Mir-21 upregulated the expression of VCAM-1 and monocyte chemotactic protein-1 (MCP-1) by suppressing the peroxisome proliferators-activated receptor-α (PPARα), increasing adhesion of monocytes to ECs and proinflammatory responses [93].

MicroRNA-92a, a member of miR-17~92 cluster, was upregulated by LSS in HUVECs [94]. The decreased expression of miR-92a was correlated to the upregulation of KLF2 and subsequent KLF2-mediated eNOS and thrombomodulin (TM) expression and NO production, all of which are considered as potent anti-thrombotic, anti-adhesive, and anti-inflammatory properties [94, 95]. Therefore, LSS-induced miR-92a inhibited KLF2-mediated eNOS and TM expression that resulted in impaired EC functions.

In contrast to LSS, high ESS (HSS) appears to be protective against atherosclerosis, but the precise functions still remain unclear [96]. Mir-10a is one of the flow-induced miRNAs in ECs and is known to be downregulated in athero-susceptible regions of the inner aortic arch and aorto-renal branches than other regions [97]. The knockout of miR-10a led to IκB/NF-κBmediated inflammation, which suggested anti-inflammatory role of miR-10a. Interestingly, two key regulators of IκBα degradation, mitogen-activated kinase kinase kinase 7 (MAP3K7; TAK1) and β-transducin repeat-containing gene (β-TRC), are putative targets of miR-10a [97]. Therefore, miR-10a contributes to anti-inflammatory endothelial phenotype through the inhibition of pro-inflammatory molecules in athero-susceptible regions.

In addition, Qin et al. found that laminar shear stress induced miR-19a that suppressed cyclin D1 expression, leading to an arrest of cell cycle at G1/S transition [98]. Similar research by Chen et al. showed that miR-101 was also induced by laminar shear stress and cell cycle arrest at the G1/S transition and suppressed endothelial cell proliferation via targeting mTOR [99].

MicroRNA-155 is upregulated by prolonged HSS in HUVECs and it modulated endotheliumdependent vasorelaxation by repressing eNOS. Therefore, the inhibition of miR-155 improved endothelial dysfunction during the development of atherosclerosis [100].

MicroRNA-21 and miR-92a were induced by both LSS and HSS. Compared with LSS-induced miR-21, HSS-induced miR-21 decreased EC apoptosis and activated the NO pathway by targeting PTEN [101]. As mentioned earlier, downregulation of miR-92a led to upregulation of KLF2 and subsequent KLF2-mediated eNOS and TM, which facilitated the maintenance of EC homeostasis and functions against inflammation and pro-atherosclerotic effects [94, 95].

#### **4.3. Angiotensin II (AngII) and miRNAs**

Angiotensin II (AngII) has been implicated in the development and progression of cardiovas‐ cular diseases such as hypertension, atherosclerosis, and restenosis after vascular injury [102]. AngII-mediated intracellular singling is activated through its G-protein-coupled AngII Type 1 receptor (AT1R). In both VSMCs and ECs, AngII leads to vascular and endothelial dysfunc‐ tions by generating intracellular reactive oxygen species (ROS) and negatively regulating NO signaling pathway, respectively [103, 104]. Zhu et al. found that miR-155, miR-221, and miR-222 were highly expressed in both HUVECs and VSMCs, and a key endothelial tran‐ scription factor for inflammation and tube formation Ets-1 was a putative target of miR-155, miR-221, and miR-222 [105]. Interestingly, miR-155 also targeted AT1R, and AngII-stimulated HUVECs upregulates Ets-1 and its downstream genes, including VCAM1, MCP1, and FLT1 [105]. These results indicated that miR-155, miR-221, and miR-222 act as negative regulators to inflammatory response of ECs caused by AngII stimulation.

### **4.4. Oxidized low-density lipoprotein (oxLDL) and miRNAs**

Oxidized low-density lipoprotein (oxLDL) induces EC death and dysfunction through the activation of NF-κB and AP-1 pathways, and it also activates pro-inflammatory response during the progression of atherosclerotic vascular diseases [2, 106]. Based on microarray and qRT-PCR analysis with oxLDL-stimulated HUVECs, Qin et al. found that four miRNAs are upregulating including miR-365 and miR-142-3p, whereas eleven miRNAs are downregulat‐ ing including miR-590-5p and miR-33a in microarray, which expression levels are validated with qRT-PCR [107]. Using computational prediction algorithms (TargetScan, microRNA.org, and MicroCosm v5), the gene ontology (GO) database (www.geneontology.org), and KEGG pathway database (www.genome.ad.jp/kegg), the authors predicted and identified the functions of miRNA-targeted genes. As a result, miR-365 has been reported to be involved in oxLDL-induced apoptosis of ECs by suppressing the anti-apoptotic protein Bcl-2 [107]. Therefore, the inhibition of miR-365 may reduce EC apoptosis and inhibit the pro-atherogenic progression caused by oxLDL.
