**9. The role of small RNAs in-stent restenosis**

Recent progress in molecular biology has resulted in development of numerous effective gene therapy methods via transferring RNA molecules for the treatment of variety diseases. There are also many studies with the purpose of prevention and treatment of vascular neointima proliferation after balloon angioplasty and stent implantation, using RNA molecules [56-58]. Recent studies suggest that microribonucleic acid-based (miRNA) are important gene regula‐ tors and seems to be suitable for the treatment of various cardiovascular diseases [56, 59]. Delivery and controlled release of miRNA through different polymeric materials to target tissues is one of the nucleic acid-based therapy approaches.

Discovered little more than decade ago, non-protein-coding RNAs are single-stranded endogenous RNAs, approximately 25 nucleotides long and they are called as miRNAs [60, 61]. They regulate gene expression negatively at the posttranscriptional level by binding to specific mRNA target, leading either to degradation or to translational target protein repres‐ sion, rarely they can promote gene expression [56, 59, 62-64]. Small interfering RNAs (siRNA) are short, double-stranded RNAs (20-25 nucleotides) that induce the degradation of target mRNA and inhibits the production of the target protein, and the procedure is called RNA silencing. Unfortunately, clinical applications of RNA interference-based therapeutics such as siRNAs and miRNAs have been limited mainly due to low intracellular delivery efficiency in vitro and in vivo. However, RNA molecules promising therapeutic potential, safe, and efficient delivery methods have to be developed for targeted controlled release. Over the last decade, there has been great effort to develop effective nonviral delivery systems for the transfection of siRNA and miRNA [60]. As it is well known, RNAs are short double-stranded molecules. Due to this reason, they have more rigid structures and inappropriate distribution, making them difficult to form stable and compact particles using a wide range of cationic condensing reagents, such as polylipids, polypeptides, and polyamines, via simple electrostatic interac‐ tions. Thus, to achieve maximum target gene silencing, improved gene carrier systems have to be prepared. Therefore, attention has become focused on development of nonviral gene delivery vectors to carry small RNA molecules to target cells [65].

Several experimental and clinical data showed that miRNAs are associated with resteno‐ sis or renarrowing of the arteries which primarily results from the proliferation and migration of VSMCs into the intima after stent implantation [66, 67]. Recent evidence by several groups has decelerated that miRNAs have an important role in prevention of atherosclerosis and restenosis [62, 63, 67-71]. In fact, knockdown of miR-21, miR-221, and miR-222 and overexpression of miR145 were found to be intimately relevant to neointi‐ mal formation after vessel injury [57, 58, 62, 71-73]. miR-21 is encoded by a single gene and autonomously transcribed from a conserved promotor that is located within the intron of the overlapping protein coding gene [73]. The oncogenic activity of miR-21 has been identified by several groups [74-76]. Besides, it has been found to play important role in proliferation of VSMCs, cardiac cell growth, and death and cardiac fibroblast functions [73]. Indeed, both basic and clinical studies have demonstrated that the overexpression of miR-21 in human reduces cardiac fibrosis and prevents vascular neointima proliferation after balloon angioplasty and stent implantation [72, 77].

Similarly, Liu et al. reported that both of miR-221 and miR-222 were recognized in rat carotid arteries after angioplasty, in which their expression was upregulated and localization in VSMCs at the injured regions of vascular walls [78]. Moreover, it was shown that the overex‐ pression of miR-221 and miR-222 decreased VSMC proliferation in vitro. Also, the knockdown of miR-221 and miR-222 in rat carotid arteries suppressed VSMC proliferation in vivo and neointimal lesion formation after angioplasty [78]. However, among miRNAs, miR-145 is the most abundant type in vascular walls [58]. In addition to these, especially both miR-143 and miR-145 are significantly expressed in vascular endothelial cells (VECs), which is able of controlling vascular neointimal lesion formation [57].

Recent studies suggest that microribonucleic acid-based (miRNA) are important gene regula‐ tors and seems to be suitable for the treatment of various cardiovascular diseases [56, 59]. Delivery and controlled release of miRNA through different polymeric materials to target

Discovered little more than decade ago, non-protein-coding RNAs are single-stranded endogenous RNAs, approximately 25 nucleotides long and they are called as miRNAs [60, 61]. They regulate gene expression negatively at the posttranscriptional level by binding to specific mRNA target, leading either to degradation or to translational target protein repres‐ sion, rarely they can promote gene expression [56, 59, 62-64]. Small interfering RNAs (siRNA) are short, double-stranded RNAs (20-25 nucleotides) that induce the degradation of target mRNA and inhibits the production of the target protein, and the procedure is called RNA silencing. Unfortunately, clinical applications of RNA interference-based therapeutics such as siRNAs and miRNAs have been limited mainly due to low intracellular delivery efficiency in vitro and in vivo. However, RNA molecules promising therapeutic potential, safe, and efficient delivery methods have to be developed for targeted controlled release. Over the last decade, there has been great effort to develop effective nonviral delivery systems for the transfection of siRNA and miRNA [60]. As it is well known, RNAs are short double-stranded molecules. Due to this reason, they have more rigid structures and inappropriate distribution, making them difficult to form stable and compact particles using a wide range of cationic condensing reagents, such as polylipids, polypeptides, and polyamines, via simple electrostatic interac‐ tions. Thus, to achieve maximum target gene silencing, improved gene carrier systems have to be prepared. Therefore, attention has become focused on development of nonviral gene

Several experimental and clinical data showed that miRNAs are associated with resteno‐ sis or renarrowing of the arteries which primarily results from the proliferation and migration of VSMCs into the intima after stent implantation [66, 67]. Recent evidence by several groups has decelerated that miRNAs have an important role in prevention of atherosclerosis and restenosis [62, 63, 67-71]. In fact, knockdown of miR-21, miR-221, and miR-222 and overexpression of miR145 were found to be intimately relevant to neointi‐ mal formation after vessel injury [57, 58, 62, 71-73]. miR-21 is encoded by a single gene and autonomously transcribed from a conserved promotor that is located within the intron of the overlapping protein coding gene [73]. The oncogenic activity of miR-21 has been identified by several groups [74-76]. Besides, it has been found to play important role in proliferation of VSMCs, cardiac cell growth, and death and cardiac fibroblast functions [73]. Indeed, both basic and clinical studies have demonstrated that the overexpression of miR-21 in human reduces cardiac fibrosis and prevents vascular neointima proliferation after

Similarly, Liu et al. reported that both of miR-221 and miR-222 were recognized in rat carotid arteries after angioplasty, in which their expression was upregulated and localization in VSMCs at the injured regions of vascular walls [78]. Moreover, it was shown that the overex‐ pression of miR-221 and miR-222 decreased VSMC proliferation in vitro. Also, the knockdown of miR-221 and miR-222 in rat carotid arteries suppressed VSMC proliferation in vivo and

tissues is one of the nucleic acid-based therapy approaches.

430 Muscle Cell and Tissue

delivery vectors to carry small RNA molecules to target cells [65].

balloon angioplasty and stent implantation [72, 77].

siRNAs mediate specific gene silencing through a highly regulated enzyme-mediated process. Nowadays, siRNAs are established as the most important biological strategy for gene silencing that includes the degradation of target mRNA and block production of the related protein [79, 80]. Yanming et al. [81] have found that siRNAs reduce neointima formation significantly as reflected by a decreased intima/media area ratio in carotid artery sections after surgical mechanical injury of the rat carotid artery. Wang et al. [59] reported that c-myc siRNA, when given immediately after the surgery, is an effective approach for the prevention of vein graft restenosis.

Usage of nanoparticle eluting stent technologies is an important approach. Walter and colleagues [16] have developed nanoparticles containing plasmid DNA-encoding sequence hVEGF-2 and explored the ability of delivery of target sequence by NP through the stent. An alternative and novel treatment strategy, acceleration of re-endothelialization via VEGF-2 gene-eluting stents, is achieved through endothelial cell proliferation by serving to activate endothelial cell proliferation pathways [16, 82].

Strategies for enhancing gene delivery and gene transfer through stents typically involve the complexations of siRNA/miRNA molecule with cationic polymers, which can be loaded on the stent surface [82, 83].

Although there are several in vitro gene therapy studies for the prevention of restenosis, a few studies with the use of miRNA/siRNA-based therapy for the treatment of cardiovascular diseases have been carried out in humans. Although in vivo studies of miRNA-based agents, conjugated to biodegradable polymers or encapsulated in nanoparticles, were promising, to date there have been a few studies consisting of miRNA-vehicle complexes to a polymer-coated stent that allow delivery of the miRNA for achieve endothelial cell proliferation by serving to activate endothelial cell proliferation pathways [62, 82].

Patil and Panyam [84] have developed nanoparticles using the biodegradable polymer, poly(d,l-lactide-co-glycolide) (PLGA), for siRNA delivery. Additionally, they have incorpo‐ rated in the PLGA matrix, a cationic polymer, polyethylenimine (PEI), to improve siRNA encapsulation in PLGA nanoparticles. The effectiveness of siRNA-loaded PLGA-PEI nano‐ particles was investigated in vitro. They have reported that PEI in PLGA nanoparticle matrix has increased siRNA encapsulation by about 2-fold and also improved the siRNA release profile. Moreover, they have observed higher cellular uptake and cytosolic delivery with the encapsulated siRNA.

In order to avoid such blockages, at the site of angioplasty or stent placement, the suppression of SMCs near the implanted stent, etc., has developed a new delivery technique for Akt1 (Akt1 is a protein that plays a key role in cellular proliferation) siRNA nanoparticles to release from a hyaluronic acid (HA)-coated stent surface. For this purpose, they have used disulfide crosslinked low molecular polyethyleneimine (PEI) (ssPEI) as a gene delivery carrier. Disulfide bonds are stable in an oxidative extracellular environment but degrade rapidly in reductive intracellular environments. They have immobilized Akt1 siRNA/ssPEI nanoparticles (ASNs) on the HA-coated stent surface. They have reported that the Akt1 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area in the ballooninjured external iliac artery in rabbits [85].

Encouragingly, the current developments in the understanding of RNAs have reveal both miRNAs and siRNAs as a potential targets for the development of new diagnostic and therapeutic strategies for the prevention of restenosis [56, 62, 63]. Therefore, attention has become focused on the development of chemically modified RNAs to cure or prevent in-stent restenosis.
