**5. Macrophage**

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

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

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].

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

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

inhibition of pro-inflammatory molecules in athero-susceptible regions.

endothelial dysfunction during the development of atherosclerosis [100].

to inflammatory response of ECs caused by AngII stimulation.

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

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

66 Muscle Cell and Tissue

The accumulation and activation of macrophages within the vascular wall are a crucial event in vascular remodeling and implicate the progression of atherogenesis, which is initiated by inflammation, a rise in circulating LDL levels, and the accumulation of oxLDL in macrophages [108, 109]. Inflammation triggers the recruitment and infiltration of monocytes and macro‐ phage in atherosclerotic lesions, continuously accumulating lipids and oxLDL [110]. The macrophages respond to various inflammatory stimuli, which lead to the changes in expres‐ sion of many genes and several miRNAs, including miR-155, miR-125a, miR-146a, and miR-146b [111, 112]. Although the microRNAs involved in oxLDL accumulation and inflam‐ matory response in macrophages are currently unclear, considering the role of miRNAs in VSMCs and ECs during arterial remodeling, the idea of miRNAs that also contribute the macrophage functions does not seem to be illogical (Figure 5) [2].

**Figure 5.** miRNAs in macrophages during vascular remodeling.

#### **5.1. miR-155**

The expression of miR-155 increased in human atherosclerotic lesions, but circulating miR-155 is known to be downregulated in patients with coronary artery diseases [113, 114]. During vascular remodeling, miR-155 is expressed in VSMCs, ECs, as well as activated macrophages [115]. The oxLDL and/or ESS promote the recruitment of monocytes into the arterial wall through ECs and then monocytes are differentiated toward macrophages and dendritic cells (DCs) [108]. MiR-155 is the most important miRNA in macrophage-mediated inflammation [116], and its expression is increased by several Toll-like receptor (TLR) ligands via myeloid differentiation primary response gene (MyD88) and/or TIR-domain-containing adapterinducing interferon-β (TRIF)-dependent signaling in the inflammatory activation of macro‐ phages [117]. In addition, the lipopolysaccharide (LPS) is also known to induce miR-155 in monocytes and DCs. LPS-induced miR-155 expression is mediated by the single-strand RNAbinding protein KH-type splicing regulatory protein (KSRP) in bone marrow-derived macro‐ phages [118]. In activated primary human monocyte-derived DCs, LPS-induced upregulation of miR-155 inhibited Toll-like receptor/interleukin-1 (TLR/IL-1) inflammatory pathway as well as TAB2 (an adaptor in the TLR/IL-1 signaling cascade), attenuating IL-1β and inflammatory signals [119]. Furthermore, oxLDL and cytokine IFN-γ induced the expression of miR-155 that modulated the inflammatory response in macrophages by repressing BCL6, a negative regulator of pro-inflammatory NF-κB signaling. The attenuation of BCL6 was correlated to upregulation of chemokine CCL2, an activator of monocyte recruiting into atherosclerotic plaques [120]. MicroRNA-155 also regulates TGF-β signaling in macrophages by targeting SMAD2. Overexpression of miR-155 inhibited TGF-β induction of SMAD2 phosphorylation, which repressed TGF-β-dependent transcription of cytokines, such as IL-1β [121].

On the other hand, a potent anti-inflammatory cytokine IL-10 inhibited LPS-induced miR-155 expression via STAT3-dependent manner, which led to upregulation of the target proteins of miR-155 such as Src homology 2 domain-containing inositol-5-phosphatase 1 (SHIP1) and antiinflammatory genes [122]. Moreover, LPS-activated PI3K/Akt1 pathway inhibited expressions of miR-155 and pro-inflammatory mediators [123]. Paradoxically, a small number of studies reported that miR-155 is also downregulated in oxLDL simulation and reduces lipid uptake and accumulation in macrophages [120, 124]. Therefore, miR-155 in macrophages maintains the balance between pro- and anti-inflammatory responses, which is influenced by the diseases context or extracellular microenvironments.

#### **5.2. miR-125a**

Similar to miR-155, upregulation of miR-125a seems to decrease lipid accumulation by directly targeting oxysterol-binding protein-like 9 (ORP9), but the functional role of miR-125a in oxLDL-stimulated monocytes and macrophage still remains unclear [125]. MicroRNA-125a has been reported to be upregulated through oxLDL in primary human monocytes, and inhibition of miR-125a increased the secretion of IL-6, TNF-α, IL-2, and TGF-β, possibly due to enhanced expression of scavenger receptors (LOX-1, CD68).

#### **5.3. miR-146a and miR-146b**

MicroRNA-146a/b is upregulated after stimulation with oxLDL in primary human monocytes [125]. MiR-146a and -b are located on different chromosomes, and differ by two nucleotides. Stimulation of TLR-2, 4, and 5 has been reported to induce miR-146a/b expressions in macro‐ phages through NF-κB activation [126]. The oxLDL-mediated suppression of miR-146a promoted lipid uptake and cytokine release, most likely due to reduced suppression of TLR-4 (target of miR-146a) in macrophage [124]. Additionally, miR-146a and b are known to upre‐ gulated in human atherosclerotic plaques [127].
