**12. miRNAs in atherosclerosis and neointimal hyperplasia**

Although roles of various miRNAs and their participation in biological processes have been recognized in various cultured cells or animal models, and expression profiles of circulating miRNAs in patients of cardiovascular diseases [50, 51], involvement of miRNAs in human atherosclerotic plaques has received little attention. However, one of the recent studies investigated miRNA/mRNA expression profiles of human atherosclerotic plaques from peripheral arteries in comparison to nonatherosclerotic left internal thoracic arteries to determine the relationship between miRNA/mRNA expression profiles and biological processes in atherosclerosis [70]. Results of this study revealed significant amounts of miRNA-21,-34a, -146a, -146b-5p, and -210 expressions in atherosclerotic lesions. Consis‐ tent with this there was downregulation of several predicted targets of these miRNAs in atherosclerotic plaques. According to the combination of miRNA/mRNA profiles and bioinformatic analysis, nine KEGG pathways including immunodeficiency, metabolism, p53 and cell proliferation signaling pathways enriched with predicted targets were significantly upregulated. On the contrary, VSMC contraction and purine metabolism were downregulated.

## **13. miRNAs in restenosis**

64]. miRNA-146a is shown to directly target Krupple-like factor-4 (KLF-4) and promote VSMC proliferation in cultured rat VSMC and vascular neointimal hyperplasia [32, 60, 65]. KLF-4 and miRNA-146a appear to exhibit a feedback relationship regulating each other's expression. While miRNA-146a inhibits KLF-4 expression by targeting the 3'-UTR region of KLF-4, KLF-4 inhibits miRNA-146a at the transcriptional level. KLF-5, another member of KLF family promoted the transcription of miRNA-146a. It appears these molecules form a regulatory

Certain miRNAs including miRNA-143, miRNA-145 and miRNA-26a alter VSMC phenotype by causing suppression of VSMC proliferation (Table 1). Among these miRNAs, miRNA-143 and -145 are considered master regulators of contractile phenotype by promoting contractile protein expression [32, 58, 60]. Moreover, miRNA-145 not only stimulates differentiation of adult VSMC, but also promotes differentiation of multipotent neural crest stem cells into VSMC [57]. In normal vessel walls the miRNA-143/145 cluster is lavishly expressed. However, both miRNAs are dramatically reduced not only in injured carotid arteries following angio‐ plasty [32, 58, 60, 66] but also downregulated in different cancer cell lines [67]. Further studies proved that miRNA-145 is a critical modulator of VSMC differentiation via its target gene KLF-5. Consistent with this, while the use of miRNA-145 oligonucleotide mimics upregulated the expression of VSMC differentiation marker genes such as SM α-actin, calponin, and SM-MHC, both at gene and protein levels, overexpression of KLF-5 reduced the gene expression of SM α-actin implicating a relationship between miRNA-145 and KLF-5 gene in VSMC

Analysis of growth arrested human aortic VSMC by miRNA array screening identified upregulation of miRNA-26a in differentiated VSMC, which is associated with reduction in SMAD activity [59, 60, 68]. This miRNA is dramatically downregulated in two murine models

Embryonic stem cells are known to differentiate to VSMC and one of the factors that induces VSMC differentiation is all trans retinoic acid, which in addition to regulating a wide variety of protein coding genes it also regulates expression of miRNAs that affect smooth muscle cell differentiation. It is found that expression of miRNA-10a contributes to retinoic acid-induced VSMC differentiation by negatively regulating its target histone deacetylase 4 [69]. Involve‐ ment of miRNAs in stem cell and vessel wall progenitor cell differentiation has significant implications in the pathogenesis of atherosclerosis, the response to vascular injury and

Recently presence of miRNAs is demonstrated in circulating blood, which may be useful as biomarkers for diseases [51]. Analysis of serum or plasma for circulating levels of miRNAs in

control to appropriately modulate VSMC proliferation [32, 60].

differentiation.

154 Current Trends in Atherogenesis

of aneurysm.

vascular remodeling.

**11. Circulating miRNAs**

**10. miRNAs in the suppression of VSMC proliferation**

Role of miRNAs in restenosis is mainly studied using the common rat carotid artery balloon injury animal model. miRNA profiles in the carotid artery is determined by using miRNA arrays [62]. One of the miRNA that was aberrantly overexpressed in injury-induced neointimal lesions is miRNA-21. miRNA-21 promotes VSMC proliferation and inhibits apoptosis of VSMC by directly targeting PTEN and programmed cell death 4, respectively [32]. Similarly miRNA-221 and -222, which are encoded by a gene cluster on X chromosome, share the same seed sequence, identical targets and similar functions were upregulated in balloon-injured carotid arteries. Consistent with their upregulation, their target genes, p27kip1 and p57kip2 were downregulated [32, 64]. Additionally, miRNA-143 and miRNA-145 that promote VSMC differentiation and expressed highly in vascular tissue, were significantly reduced in apoli‐ poprotein E knockout mice where vascular injury was induced by hypercholesterolemic diet [71]. Cooperatively, both miRNA-143 and miRNA-145 target a network of transcription factors such as Elk1, KLF-4 and myocardin to stimulate differentiation and inhibit proliferation of VSMC. Taken together, these studies indicate significant role of miRNA-143/miRNA-145 in VSMC differentiation and vascular disease.

The miRNA-17-92 cluster is a polycistronic miRNA gene, which is titled as oncomir-1 in humans because of their oncogenic properties and overexpression in different cancers [79]. The miRNA-17-92 primary transcript encodes six mature miRNAs: miRNA-17,-18a, 19a, 20a, 19b-1, and 92a-1 that are tightly grouped within an 800 base-pair region of human chromosome 13 [80]. For some of these members corresponding target genes have been identified, which include cell cycle inhibitor CDKN1A (p21Cip1) and pro- apoptotic PTEN and BCL2L11 (Bim). Furthermore, transcription of miRNA-17-92 has been shown to be activated by c-myc tran‐ scription factor [78]. In our earlier studies butyrate has been shown to downregulate c-myc [81] and upregulate CDKN1A (p21Cip1) [72-75] in proliferation inhibited VSMC. Based on these observations, it appears by downregulating c-myc expression potentially via epigenetic modification, butyrate inhibits expression of miRNA-17-92 cluster with a corresponding increase in miRNA-17-92 target genes such as CDKN1A (p21Cip1). Taken together, our preliminary miRNA expression data emphasizes role of miRNAs in antiproliferative and chemoprotective effects of butyrate in VSMC. Further studies are under investigation to confirm the role of miRNA-17-92 cluster in the regulation of VSMC proliferation by investi‐ gating the effects of miRNA mimics of miRNA-17-92 cluster in reversing the effect of butyrate on VSMC proliferation and on decreasing the levels of their target proteins. Utilization of this information is beneficial in targeting miRNAs aimed to decrease the level of pathogenic/ aberrantly expressed miRNAs or to increase miRNAs with valuable functions in the interven‐

MicroRNAome of Vascular Smooth Muscle Cells: Potential for MicroRNA-Based Vascular Therapies

http://dx.doi.org/10.5772/54636

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**15. miRNAs as new therapeutic targets for vascular proliferative diseases**

Despite the substantial progress in understanding the etiology and clinical management of vascular proliferative diseases, they are still life threatening diseases responsible for the global burden of cardiovascular diseases. Clinically, medications and surgical procedures are the only methods of treatment for patients with atherosclerotic disease. Atherosclerotic patients are generally treated by angioplasty with stent replacement but it commonly leads to restenosis in significant number of angioplasty patients. Phenotypic modification of VSMC from contractile differentiated state to proliferative dedifferentiation state is the primary pathophy‐ siological mechanism in the development of atherosclerosis and in different clinical patholo‐ gies such as postangioplasty restenosis, in-stent restenosis, vein bypass graft failure and transplant vasculopathy [33,34]. Therefore, understanding the molecular mechanisms of VSMC proliferation may offer novel insights into disease pathogenesis leading to targeted therapies. Vascular phenotypic modulation is a multifactorial process involving multiple pathways and multiple genes. Based on the current understanding of the roles of miRNAs in the normal development and in disease pathogenesis, it appears miRNA-based therapy has a potential in vascular proliferative diseases, particularly because one endogenous miRNA can target its multiple target genes. Moreover, demonstration of changes in expression of certain miRNAs that is specifically associated with particular VSMC phenotype in different models of studies, as depicted in this article, clearly suggests that expression analysis of miRNA will provide insights into vascular proliferative disease mechanisms and possibly identifies novel

tion of occlusive vascular proliferative diseases.
