Combined Roles of miR-126-3p and miR-126-5p in Endothelial Regeneration and Their Use in Therapeutic Approaches

miR-126, the highest miRNA expressed in ECs, is encoded by exon 7 of the Egfl7 gene (Fish et al. 2008; Wang et al. 2008). Out of the miR-126 precursor, two mature miRNA strands are generated, miR-126-3p (guide strand) and miR-126-5p (passenger strand). The key role of the miR-126-3p in the vasculature was highlighted in studies with genetic deletion or inhibition of this strand. miR-126-3p regulates vascular integrity and angiogenesis by targeting Spred-1 and PIK3R2 as determined in mouse and zebrafish, respectively (Wang et al. 2008; Fish et al. 2008). In addition, miR-126-3p regulates vascular inflammation by targeting VCAM1 in ECs (Harris et al. 2008). Notably, the expression of miR-126-5p is upregulated in vascular precursor cells and ECs during embryogenesis, and its expression can be even higher than the expression level of miR-126-3p (Fish et al. 2008; Neth et al. 2013). Moreover, miR-126-5p expression levels are comparable to that of miR-126-3p in various adult mouse tissues (Schober et al. 2014). This data limelights that both mature miRNA strands generated from miR-126 are functional in endothelial cells.

Indeed, miR-126-5p plays a key function in EC proliferation improving reendo- thelialization after vascular injury by targeting Delta-like 1 homolog (DLK1) (Schober et al. 2014). DLK1 is a noncanonical inhibitor of NOTCH1 highly expressed in various tissues during development and is downregulated in most tissues after birth (Rodriguez et al. 2012; Baladron et al. 2005; Falix et al. 2012). Interestingly, DLK1 is upregulated following vascular injury and counter-regulates increased EC proliferation during endothelial repair probably by targeting the NOTCH1 receptor (Rodriguez et al. 2012; Schober et al. 2014). These effects in the modulation of reendothelialization in injured arteries are exclusive of miR-126-5p indicating functional specialization of the individual miR-126 strands (Schober et al. 2014). The guide strand miR-126-3p supports endothelial recovery promoting the angiogenic growth factor signaling through targeting of Spred1 (Jansen et al. 2013).

Endothelial proliferation is rare in the adult vasculature; however, increased EC turnover occurs at predilection sites of atherosclerosis in response to increased levels of stress-induced EC death and detachment (Bartling et al. 2000; Zeng et al. 2009; Foteinos et al. 2008). Notably, miR-126-5p, but not miR-126-3p, is down- regulated at predilection sites characterized by a disturbed flow, probably due to differences in the stability and processing of the individual miR-126 strands (Schober et al. 2014). The reduced miR-126-5p levels at predilection sites upregu- late DLK1 expression, thereby controlling and limiting EC proliferation (Schober et al. 2014). Laminar blood flow not only protects EC functions and limits EC turnover but also provides a higher proliferative capacity by inducing miR-126-5p expression at non-predilection sites (Schober et al. 2014).

The prevalence of endothelial ER stress and enhanced EC apoptosis are characteristics of predilection sites (Zeng et al. 2009; Civelek et al. 2009). During atherosclerosis, modified LDL induces EC apoptosis by activating ER stress via LOX-1 receptor (Hong et al. 2014). Under this stress conditions, resident ECs surrounding the damaged regions proliferate in order to heal the damaged part (Itoh et al. 2010). Modified LDL has been shown to inhibit EC replication in vitro and hyperlipidemia significantly reduces EC proliferation at predilection sites in vivo (Schober et al. 2014; Chen et al. 2000). Under these hyperlipidemic conditions, the DLK1-mediated antiproliferative response becomes detrimental at the predilection sites and results in impaired EC regeneration. At the non-predilection sites, however, the high levels of miR-126-5p suppress DLK1 expression thereby enabling EC regeneration in response to hyperlipidemia (Schober et al. 2014). Consequently, a replenishment of the miR-126-5p pool by an exogenous therapy with mimics under hyperlipidemic condition improves EC proliferation at predilection sites and limits atherogenesis (Schober et al. 2014). Hence, the miR-126-5p strand plays an atheroprotective role in response to hyperlipidemic stress by maintaining the endothelial proliferative reserve through targeting DLK1 (Schober et al. 2014). Similarly, the restoration of other miRNAs have been shown to also be an adequate strategy in atherosclerosis. Hyperlipidemia downregulates also miR-181b crucial for the inhibition of the NF-кр signaling in endothelial cells; thus, using exogenous miR-181b helps to reduce atherosclerosis (Loyer et al. 2014; Sun et al. 2014). Conversely, sometimes the inhibition of miRNAs is beneficial. During the atheroprotective laminar flow, the levels of miR-92a are decreased, which in turn increases KLF2 expression to maintain endothelial homeostasis (Loyer et al. 2014; Sun et al. 2014). Indeed, large- scale miRNA profiling in endothelial cells identified miR-92a as a candidate, which is preferentially upregulated by the combination of disturbed flow and oxidized LDL through STAT3, as confirmed in atheroprone regions and mouse models (Loyer et al. 2014; Sun et al. 2014). Specific in vivo blockade of miR-92a expression targeting Socs5 reduced endothelial inflammation, decreased atherosclerotic plaque size, and promoted a more stable lesion phenotype. Hence, miR-92a antagomirs may serve as another atheroprotective therapeutic strategy.

Besides its functions in EC proliferation, miR-126-5p promotes also leukocyte adhesion by targeting SetD5 and represses transendothelial migration by degrading ALCAM in retinal and pulmonary ECs, respectively (Poissonnier et al. 2014). However, it seems that this is not involved in atherosclerosis as miR-126 mature strands inhibit the recruitment of inflammatory monocytes to the tumor microenvironment (Zhang et al. 2013). Thus, the miR-126-5p strand can regulate crucial cell functions such as proliferation and inflammation depending on the transcripts targeted and the tissue where it is present.

Both miR-126 mature strands, miR-126-5p and miR126-3p, act synergistically for the preservation of vasculature. Whereas miR-126-5p protective activities are principally intracelullarly mediated, miR-126-3p needs to be release for its atheroprotective activities. miR-126-3p is delivered to vascular cells via endothelial- derived apoptotic bodies, reducing atherosclerosis through the repression of RGS16 that leads to an upregulation of CXCL12 expression (Zernecke et al. 2009). The chemokine CXCL12 not only triggers the recruitment of angiogenic progenitor cells to the atherosclerotic sites but also increases its own mRNA in an autoregulatory loop thereby also supporting endothelial function (Akhtar et al. 2013; Zernecke et al. 2009; Kuhlmann et al. 2005; Wei et al. 2013b). This system of atheroprotection appears to be disrupted upon disease, and so circulating levels of miR-126-3p has been detected to be substantially reduced in patients with coronary artery disease or insulin resistance/diabetes (Fichtlscherer et al. 2010; Zampetaki et al. 2010).

In addition to the vesicle-mediated transfer, miR-126-3p is transmitted by Ago2 protein from ECs to SMCs, where it targets Foxo3, Bcl2, and Irs1 thereby

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increasing SMC turnover (Zhou et al. 2013). These data indicate that miR-126-3p is mainly transferred to the vascular cells and improves cellular regeneration while miR-126-5p stays within ECs and controls endothelial regeneration. Understanding the functional roles of miR-126 mature strands in regulation of endothelial cell function provides new insights on complimentary regulatory mechanisms mediated by miRNA pairs in atherosclerosis (Fig. 1.3).

However, the mechanisms determining differential stability and trafficking of sister strands are not fully understood. It needs to be further studied how one strand of a miRNA duplex can be tagged as export good preferentially leaving the cell, whereas the antisense strand is rather maintained in the cell.

Complementary role of miR-126-3p/5p pair on vascular EC homeostasis during atherosclerosis

Fig. 1.3 Complementary role of miR-126-3p/5p pair on vascular EC homeostasis during atherosclerosis. miR-126-5p levels are sensitive to blood flow and regulate the proliferative capacity of endothelial cells through degradation of DLK1 a noncanonical inhibitor of NOTCH1, subsequently improving cell regeneration. This mechanism becomes detrimental under hyperlipidemic conditions where oxidized LDL strongly impairs endothelial cell (EC) proliferation. At the regions of laminar flow (left side of the illustration), the high levels of miR-126-5p are still capable to maintain EC regenerative potential, whereas the low levels of miR-126-5p at the predilection sites (right side of the illustration) are not sufficient to counteract this process. As a last atheroprotective effort, the apoptotic ECs under disturbed flow and hyperlipidemic conditions release miR-126-3p via apoptotic bodies to neighboring cells which may support EC regeneration at predilection sites

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