**6. Circulating miRNAs as biomarkers**

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].

context or extracellular microenvironments.

gulated in human atherosclerotic plaques [127].

to enhanced expression of scavenger receptors (LOX-1, CD68).

**5.2. miR-125a**

68 Muscle Cell and Tissue

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

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

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

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‐

In addition to the significance of miRNAs regulating gene expression at the intracellular regions, some miRNAs have been discovered in the extracellular body fluids including serum/ plasma and bloodstream [128, 129]. Interestingly, the extracellular circulating miRNAs show remarkably stable and resistant against ribonucleases, freezing, boiling, low or high pH, and so on, which signifies that some protective mechanisms can countervail degradation [130– 132]. The mature miRNAs are released from intracellular to extracellular environment via the complex with RNA-binding proteins such as Argonaute-2 (Ago2), binding to high-density lipoprotein (HDL), and loading into microvesicle bodies such as microvesicles, exosomes, or apoptotic bodies (Figure 6) [133–136].

**Figure 6.** The cellular release mechanisms of miRNAs.

Although the cellular origin and function of the circulating miRNAs remain uncertain, their expression profiles are changed by fluid types and physiology or pathology conditions [137, 138]. The extraordinarily stable and tissue/diseases specific profiles of circulating miRNAs have been considered as promising biomarkers for diagnosis of diseases [139, 140].

Several studies suggest that circulating miRNAs may be taken into the recipient cells and regulate target gene expression [141–143]. In vascular diseases, a few studies showed that circulating miRNAs modulate the function of ECs [144, 145]. Zhang et al. found that the secretion of microvesicles containing miR-150 from human monocytic cells THP-1 is increased in the plasma of patients with atherosclerosis. Secreted monocytic miR-150 is transported into human microvascular endothelial cells (HMEC-1) and then promotes HMEC-1 migration via downregulating the miR-150 target gene c-Myb [144]. Likewise, Zernecke et al. showed that EC-derived apoptotic bodies, containing miR-126, upregulates the production of CXC chemokine CXCL12, which promotes the recruitment of Sca-1+ progenitor cells and reduces the atherosclerotic lesion formation in ApoE–/– mice. These results suggest that circulating miR-126 in apoptotic bodies may have protective potential against atherosclerosis [145].
