**3. miRNAs**

(miRNAs), PIWI-interacting RNAs (piRNAs) and small nucleolar RNAs (snoRNAs); long ncRNAs (lncRNAs) that are longer than 200 nucleotides; and macro ncRNAs, longer than 200 nucleotides that can reach 100 kilobases (kb) longer without being processed into small ncRNAs [1,7,12,18]. Based on where they are derived from within the genome, lncRNAs can be distinguished from each other. There are intronic lncRNAs (transcribed between exons of genes), intergenic lncRNAs (transcribed from the space between two genes) and lncRNAs that are derived from the regions that overlap both exon and intron of a coding gene. Furthermore, each of these ncRNAs may also be in the sense or in the antisense direction. According to functional significance, ncRNAs can be divided into: (1) housekeeping ncRNAs and (2) regulatory ncRNAs. Housekeeping ncRNAs include constitutively expressed ncRNAs that are crucial for the normal function and cellular viability, which include transfer RNAs, ribosomal RNAs, small nuclear RNAs, and snoRNAs [18]. On the contrary, regulatory ncRNAs or riboregulators include ncRNAs such as miRNAs and lncRNAs that are expressed in response to external signals, during different cellular states such as cellular differentiation or at certain stages of development, influencing the expression of other genes at transcription and transla‐ tional levels [1, 7, 12, 18]. Regarding ncRNAs that are associated with transcription start sites of genes, there are different classes of ncRNAs such as promoter-associated small RNAs (PASRs) [16], transcription start site-associated RNAs (TSSa-RNAs) [19], promoter upstream transcripts (PROMPTs) [20] and transcription initiation RNAs (tiRNAs) [21]. Even though their functional roles are poorly delineated, perhaps they have a regulatory role in transcription. Among the ncRNAs, the most widely studied and comparatively well delineated regarding their functional relevance to normal development and physiology, and to pathogenesis of disease are, small microRNAs [1, 12, 22-25]. miRNA deficiencies or surpluses have been correlated with diverse clinically important diseases including various types of cancers, neurological diseases, metabolic diseases, cardiovascular diseases, and many others [22, 25-32]. Here, we provide an overview of the current knowledge of miRNAs that participate in the regulation of vascular smooth muscle cells (VSMC) phenotypic modulation and present the potential opportunities for miRNA-based therapeutic and diagnostic approaches for vascular proliferative diseases due to atherosclerosis and restenosis. Finally, we briefly describe our preliminary unpublished data on miRNA expression profile of VSMC in response

Vascular cell activation and remodeling are the principle events in vascular pathologies such as atherosclerosis, transplant vasculopathy, post angioplasty restenosis, in-stent restenosis and bypass graft failure [33, 34]. It is realized that injury to vessel wall by various atherogenic insults sets-off inflammatory response causing endothelial cell dysfunction. Following endothelial cell dysfunction, VSMC in the media that are quiescent and contractile in nature, migrate to intima in response to local inflammation and become proliferative cells. VSMC are highly specialized cells whose principal function is to regulate the attributes of blood vessels in the body by appropriately responding to changes in the volume of blood vessels and the

to butyrate, a histone deacetylase (HDAC) inhibitor.

**2. Atherosclerosis and restenosis**

148 Current Trends in Atherogenesis

miRNAs are endogenous, well conserved, small ncRNAs, usually 20 to 26 nucleotides, that mediate posttranscriptional gene silencing by complimentary binding to the 3'-untranslated region (3'-UTR) of their target mRNA, leading to direct target mRNA degradation or transla‐ tional repression, a key phenomenon for controlling gene expression in a tissue- and devel‐ opment-specific manner [1, 38-40]. They were first detected in Caenorhabditis elegans as regulators of development in 1993 [41] and since then they have been found in many species of plants and animals. There are several differences between plants and eukaryotic mRNAs. In plants, transcriptional repressions require a perfect or near-perfect target match, whereas mismatched target can cause gene silencing at the translational level in eukaryotes. In eukar‐ yotes, miRNA complementarity typically includes the 5' bases 2-7 of the miRNAs, which is referred as miRNA "seed" region, Furthermore, one miRNA can target many different sites on the same mRNA or many different mRNAs, and a single mRNA can be under stringent but redundant control of several miRNAs. Another difference is the location of target sites on mRNAs. In eukaryotes miRNA target sites are in the 3'-UTRs of the mRNAs. In plants, target sites are normally in the coding region but they can be present in the 3'-UTRs.

(poly A) tail on 3' end [55]. Following transcription by RNA polymerase II/III, pri-miRNA transcripts are trimmed to about 60 to 100 nucleotide hairpin structures with ~2 nucleotide 3' overhang to form precursor miRNAs (pre-miRNAs) by the action of nuclear microprocessor complex. Microprocessor complexes are formed of Drosha (RNASEN), a nuclear ribonuclease RNase III enzyme and its partner DGCR8 (DiGeorge critical region 8) also called as Pasha (Partner of Drosha). The pre-miRNA transcripts are then shuttled to cytoplasm for further processing via Exportin5 and Ran-GTP6. Pre-miRNAs are processed further in the cytoplasm by the action of Dicer, another RNase III enzyme, with the assistance of double-stranded RNA binding proteins (dsRBPs) including TRBP (tar RNA binding protein), resulting in the cleavage of hairpin loop of pre-miRNAs leading to formation of ~22 nucleotide mature miRNA duplexes. Mature duplex is composed of a matured miRNA strand referred as guide strand and a complimentary strand referred as the passenger strand. The gene silencing capability depends on Dicer-mediated loading of one of the miRNA strands, usually guide strand, in the RNA-induced silencing complex (RISC) together with Argonaute (Ago) protein. The RISC guides the miRNA to bind to its complementary sequence within the 3'-UTR of its target mRNA. The degree of complementarity between the seed sequence of the miRNA and the 3'- UTR of its target mRNA determines whether to mediate mRNA degradation or to disrupt

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

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

151

miRNAs are relatively new regulatory molecules that are identified about a decade ago and demonstrated to have regulatory role in every organism and in every biological functions influencing normal biology and disease process. Once again, oncology research is in the leading position in understanding miRNA involvement in human diseases. Although most of the miRNA knowledge is coming from cancer research, during the past few years their role in other systems and diseases are emerging and rapidly being evaluated with new technologies such as deep sequencing. It is not surprising that interest in miRNA is also on the raise in cardiovascular research field. Literature on the roles and functions of miRNAs in normal cardiovascular development and in vascular pathologies is escalating [32, 46, 56-60]. Further‐ more, importance of miRNAs in the regulation of VSMC development and phenotypic modification, and response to injury is swiftly being explored because VSMC proliferation and migration are important events in vascular proliferative diseases. Here we will summarize recent updates on the significance of miRNAs in VSMCs and their role in phenotypic modu‐ lation of VSMC, thus to vascular proliferative diseases [32, 57-60]. Most of the knowledge of VSMC miRNAs is coming from culture cells, animal models and blood samples of cardiovas‐

Because activity of Dicer is essential for the miRNA processing, loss of Dicer activity should result in global loss of miRNAs. Importance of miRNAs for VSMC development and biolo‐

translation.

**6. MicroRNAome of VSMC**

cular disease patients.

**7. Evaluation of essential role of miRNAs in VSMC**

miRNAs are predicted to target about 60% of protein coding transcripts [12, 42, 43]. At present the number of miRNA sequences deposited in miRBase (Release 16) include over 15,000 miRNA loci, expressing over 17,000 distinct mature miRNA sequences from 142 species [44]. Moreover, recent appreciation in miRNA research in eukaryotes implicates that these key gene expression regulators control various biological processes as diverse as cell proliferation, cell differentiation, apoptosis, and stem cell division particularly in mammalian development [38-40, 45]. In spite of tremendous advances in miRNA research, the role of miRNAs in physiological and pathophysiological processes is just emerging. Recent miRNA expression studies demonstrate miRNAs in cardiovascular development [46], brain development [47], viral infection [48], metabolism [29], different types cancer, neurologic and cardiovascular diseases [22, 25-32] suggesting link between miRNAs and wide range of tissue development and diseases. In effect, miRNAs are considered as *trans*-acting gene regulatory molecules, similar to and as important as transcription factors in the control of gene expression [49]. Although miRNAs are considered to act as intracellular RNAs to control gene expression at posttranscriptional level, recent studies have detected miRNAs in circulating blood and in cell culture medium indicating they may be useful as biomarkers of disease [50, 51].
