**4. Roles of ncRNAs in Vascular Biology and Disease**

The vessel wall is composed of endothelial cells (ECs) and smooth muscle cells (SMCs) that play central roles in vascular biology and disease. In fact, these cells can undergo profound changes in phenotype during vascular injury and remodeling; these changes are correlated with pathologies such as atherosclerosis and proliferative thickening of the vessel known as restenosis. Atherosclerosis is a chronic inflammatory disease of the arterial wall and is the major cause of death in western countries [37]. It is a complex process involving multiple cell types and the interactions of many different molecular pathways. The events that lead to the formation of atherosclerotic lesions include modification of endothelial cell function, monocyte adherence and entry into vessel wall, phenotypic modulation of smooth muscle cell, and platelet adhesion and aggregation [38]. Phenotypic modulation of smooth muscle cells is, also, crucial in the neointimal lesion formation after stent implantation [39]. Numerous ncRNAs, especially microRNAs, have been shown to govern these processes during vascular disease. In fact, miRNA control endothelial cell and vascular smooth muscle cell biology, and thereby regulate the progression of vascular disease, such as atherosclerosis and restenosis. Current evidence also suggests that other ncRNA classes, such as lnc-RNA molecules play a critical role in endothelial and smooth muscle cell function. Figure 1 summarizes the role of ncRNA classes in different cells of the vessel wall.

#### *4.1. microRNAs in Endothelial Biology and Dysfunction*

In endothelial cells (ECs) the action of specific miRNAs is important for vascular signaling and function. Different studies indicate that the major miRNA-regulating enzymes, Dicer and Drosha, are essential for angiogenic functions of endothelial cells [40,41]. The endothelial-specific miR-126 is the most abundant miRNA found in adult ECs and it is involved in endothelial dysfunction and inflammation [42]. It is interesting to observe how miR-126 regulates the response of ECs to VEGF by inhibiting sprout-related protein SPRED1, a negative inhibitor of VEGF signaling [43]. Another group demonstrated that VCAM-1 is a direct target of miR-126 [44]. In the early phase of atherosclerotic disease, inflammatory cytokines increase a series of adhesion molecules, such as VCAM-1, on the surface of ECs. Inhibition of miR-126 increases leukocyte adherence in TNFα-stimulated ECs. Endothelial cell functions are critically regulated by other microRNAs: the miR-17-92 cluster, a polycistronic miRNA gene that produces six mature miRNAs: miR-17, miR-18a, miR-19a, miR-19b-1, miR-20a, and miR-92a [45]. Individual members of the miR-17-92 cluster, function as negative regulators of angiogenesis. In particular, miR-92a inhibited angiogenesis by targeting several functional genes, including integrin α5 (ITGa5) [46]. In addition, miR-92a negatively regulates KLF2 and KLF4 expression in athero-susceptible endothelium [47]. Given that both endothelial KLF4 and KLF2 are implicated in protection against atherogenesis [48–50], miR-92a may be important in arterial disease. Moreover, we have recently analyzed the effect of miR-92a in endothelial cell by loss-of-function studies [51]. Our group demonstrated that systemic administration of a complementary oligonucleotide (antagomiR-92a) significantly enhanced re-endothelialization in carotid arteries after balloon injury or arterial stenting. Our group and others [46,51] showed the relationship between miR-92a and endothelial nitric oxide synthase (eNOS) expression. Nitric oxide (NO) limits the formation of neointimal hyperplasia in animal models of arterial injury to a large part by inhibiting vascular smooth muscle cell proliferation [52]. Accordingly, the functional consequences of the miR-92a inhibition are an increase in NO bioavailability and an antiproliferative effect on SMCs [51]. A further example of negative correlation between microRNAs and eNOS activity is represented by miR-221 and miR-222. These microRNAs are highly expressed in ECs and exhibit anti-angiogenic effects [53]. Notably, over-expression of miR-221 and miR-222 indirectly reduces the expression of eNOS [33]. miR221 and miR-222 directly target c-kit, the receptor for stem cell factor (SCF), which plays a key role in endothelial cell migration [53]. Recently it has been shown that the miR-221 and miR-222 are negatively correlated with the expression of Ets-1 [54] that regulates the expression of several inflammatory molecules in the endothelial cell during vascular inflammation [55]. Another miRNA which has been identified in endothelial cells is miR-155. Similar to miR-221 and miR-222, miR-155 directly targets ETS-1 in ECs [54]. Also, miR-155 down-regulates eNOS expression through decreasing eNOS mRNA stability by binding its 3'-UTR [56]. Given their role in regulating endothelial cell biology, miR-221, miR 222 and miR-155 represent possible therapeutic targets in the inflammatory response of endothelial cells during the initial stage of atherosclerosis. Several other groups provide additional examples of the intersection between microRNAs and endothelial cell activation and dysfunction. In response to inflammatory stimuli, the nuclear factor-KappaB (NF-κB) signaling pathway is activated leading to the expression of multiple pro-inflammatory genes in ECs [57]. In fact, in Apolipoprotein E (ApoE)-deficient mice, endothelial cell-specific inhibition of NF-κB resulted in reduced development of atherosclerosis [58]. Two endothelial-specific microRNAs, miR-10a and miR-181b, inhibit the activation of the NF-κB signaling pathway in ECs. Recently, miR-181b has been identified as a key player in vascular inflammatory disease. miR-181b expression is reduced in response to TNF-a in the vascular endothelium, whereas its over-expression inhibits TNF-α-induced NF-κB-responsive targets gene such as VCAM-1 and E-selectin [59]. Moreover, miR-181b targets importin-α3, a critical protein in NF-κB nuclear translocation and activation. miR-10a directly inhibits mitogen-activated kinase kinase kinase 7 (MAP3K7) and beta-transducin repeat-containing gene (β-TRC) [60]. These molecules are essential in promoting IκBα degradation, an inhibitor of NF-κB activation. Inhibition of miR-10a enhances the NF-κB-dependent expression of adhesion molecules in ECs. Other specific microRNAs that regulate endothelial cell function have been described. For example, miR-125a-5p and miR-125b-5p have been identified as negative regulators of ET-1 [61], a potent vasoconstrictive and mitogen peptide that plays multiple roles in the progression of vascular disorder [62]. Taken together, the results described above indicate that several microRNAs play an essential role in endothelial pathophysiology. Accordingly, the identification of specific microRNAs involved in biological processes, such as angiogenesis and inflammation, could lead to the definition of new strategies to treat vascular diseases. Given that the same microRNAs may have opposite effects in different biological contexts, further studies are necessary to clarify their roles in endothelial dysfunction. For example, identification of signaling pathways which modulate the activity of microRNAs is critical for development of microRNA-based therapeutic strategies.

### *4.2. microRNAs in Phenotypic Switching of VSMCs*

SMCs within adult animals retain remarkable plasticity and can undergo profound and reversible changes in phenotype, a process referred to as phenotypic switching [63]. SMCs play a role during all phases of the atherogenic process as well as in proliferative disease [64–66]. Several microRNAs are implicated in VSMC phenotypic switching in response to vascular injury or atherosclerotic disease.
