Vascular miRNAs After Balloon Angioplasty

Vascular miRNAs After Balloon Angioplasty Alberto Polimeni, Salvatore De Rosa, and Ciro Indolfi*

MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression through translational repression or degradation of the target mRNA. Available strategies to improve stent patency lead to the risk of potential stent thrombosis. Modulation of miRs could be a promising means of reducing VSMC proliferation while increasing endothelial regeneration at the same time. Therefore, the goal of this review is to summarize recent experimental evidences on the role played by miRNAs in vascular remodeling, and particularly on VSMC phenotype switch and in endothelial cells, in response to vascular injury. (Trends Cardiovasc Med 23:9-14) C 2013 Elsevier Inc. All rights reserved.

• Introduction Arterial remodeling is a dynamic process and includes both physiological adaptive responses and the abnormal reactive phenomena (Indolfi et al. 1995a, 2003). Examples include but are not limited to atherosclerotic lesion formation and progression, wall hypertrophy during arterial hypertension and restenosis after percutaneous vascular interventions, especially when a stent is implanted. In fact, even though some degree of neointimal proliferation is required to warrant proper healing after endovascular stent implantation, excessive neointimal formation often follows the mechanic injury, leading to lumen narrowing (Indolfi et al. 2000, 2002).

Alberto Polimeni, Salvatore De Rosa, and Ciro Indolfi are at the Division of Cardiology, Magna Graecia University, URT Consiglio Nazionale delle Ricerche (CNR), Catanzaro 88100, Italy. *Address Correspondence to: Prof. Ciro Indolfi, Department of Medical and Surgical Sciences and URT Consiglio Nazionale delle Ricerche (CNR), Magna Graecia University, Catanzaro 88100, Italy. Tel.:þ39 09613647151; fax:þ39 09613647153; e-mail: [email protected]. & 2013 Elsevier Inc. All rights reserved. 1050-1738/$-see front matter TCM Vol. 23, No. 1, 2013

Vascular smooth muscle cells (VSMCs) and endothelial cells (ECs) are the main actors within these processes, also gathering a number of stimuli coming from other cell populations, such as monocytes/macrophages, platelets and cells of the immune system (Casscells 1992). In fact, neointimal hyperplasia is increased after vascular injury in high-risk conditions, such as diabetes (Indolfi et al. 2001a). Researchers are currently putting a lot of effort in finding an optimal strategy to reduce VSMC proliferation without affecting (or increasing) endothelial regeneration after stenting, in order to avoid the catastrophic consequences of late thrombosis associated with drug-eluting stent implantation. Recent evidences demonstrate that all the above-cited processes are thoroughly regulated by microRNAs (miRNAs)—a novel class of endogenous, short, non-coding RNAs that negatively regulate gene expression through posttranscriptional repression or degradation of specific target miRNAs (Bartel 2004). A single miRNA is able to regulate the expression of multiple genes. Altogether, miRNAs are thought to regulate at least 30% of all genes, being involved in the regulation of all major cellular functions. In addition, miRNAs have

numerous targets often within the same biological pathway. Moreover, multiple miRNAs can target the same gene and interact with each other, forming miRNA– miRNA cotargeting networks (Zampetaki et al. 2012). As they can be actively exported or uptaken by cells, miRNAs can cross cellular borders and exert their function in other cells (Hergenreider et al. 2012). MiRNAs are highly expressed in the vascular wall (Figure 1) and have emerged as key regulators of both homeostatic and pathologic processes. As such, they not only are good candidates as novel biomarkers, but also represent potential therapeutic targets. The goal of this review is to summarize recent experimental evidences, including new data from our laboratory, on the role of miRNAs in vascular remodeling. In particular, we focus on the regulation of neointimal formation and vascular response to injury.

• MiRNAs Regulate VSMCs and ECs After Vascular Injury VSMC phenotype is tightly regulated, since it plays a pivotal role in neointimal formation, constantly oscillating between a contractile and a synthetic status, which is characterized by a lower content in contractile protein. A number of molecular pathways are involved in the modulation of VSMC phenotype switch, including the platelet-derived growth factor (PDGF) and transforming growth factor (TGF)b/bone morphogenetic protein (BMP) pathways. In particular, PDGF signaling induces a proliferative phenotype, while the activation of TGF-b/BMP pathways promotes a contractile status (Chan et al. 2010). We have previously shown the pivotal role of the Ras–MAPKKs proteins and the cAMP–PKA signaling in regulating the response of VSMC to vascular injury (Indolfi et al. 1995b, 1997, 2001b). Figure 2 depicts the most important regulatory pathways involved in VSMC phenotype switch. With the advent of coronary stenting, VSMC proliferation was shown to be solely responsible for in-stent restenosis (Curcio et al. 2011). Consequently, the introduction of drug-eluting stents (DESs) has dramatically reduced coronary restenosis to less than 10% in several randomized trials 9

arteries. Among these, several miRNAs have been shown to be dynamically regulated after vascular injury. Table 1 reports the most important miRNAs, which are regulated in response to injury, with the relative targets and the net biological effect.

MiRNAs and Pro-contractile Phenotype

Figure 1. Expression of miRNAs within the vascular layers with relative biological function.

Figure 2. Regulation of vascular smooth muscle cell (VCMS) phenotypic switch, including miRNAs target knots. VSMC phenotype is tightly and dynamically regulated in response to several external stimuli, alternating between a ‘‘contractile’’ and a ‘‘synthetic’’ phenotype state. Deregulation of switching between these phenotypes is associated with vascular disorders and atherosclerosis. In normal condition, plateletderived growth factor (PDGF) induces multiple aspects of the synthetic VSMC phenotype, including augmented proliferation and migration. Inversely, the transforming growth factor-b (TGF-b) and the related factor bone morphogenetic protein 4 (BMP-4) reduce VSMC proliferation. Recent studies have highlighted the key role exerted by miRNAs in modulating VSCM development and phenotype switching. The most important intersection points between ‘‘classical’’ signaling pathways regulating VSMC phenotype switch and miRNAs are reproduced in the figure.

(Indolfi et al. 2005). Unfortunately, drugeluting stents inhibit both VSMC and EC. In this context, microRNAs could represent a novel strategy to interfere with neointimal formation and fostering, at the 10

same time, endothelial regeneration after vascular injury. Using a microarray approach, Ji et al. (2007) reported a number of miRNAs to be highly expressed in normal carotid

The miR-143/145 cluster is an important regulator of VSMC, representing the prototype regulatory system for VSMC phenotype regulation. In fact, the key role played by miRNAs in modulating VSMC phenotype was first reported in studies on differentiation of multipotent stem cells into VSMC, which was mediated by the miR-143/145 cluster (Cordes et al. 2009). MiR-145 is highly represented in the vascular wall, and its up-regulation is responsible for the induction of key genes involved in vascular differentiation. Conversely, interference with the miR-145 has an opposite effect (Cheng et al. 2009). Further evidence shows that miR-145 mediates phenotypic modulation of VSMC through its target gene KLF5 and its downstream signaling molecule, myocardin, as well as KLF4 and calmodulin kinase II delta (CamkIId) (Cordes et al. 2009). The miR-143 is also highly expressed in muscularized arteries and exerts a key regulatory function on proliferation and contractile function in VSMC, by targeting Elk-1 and ACE (Cordes et al. 2009). In line with these evidences, it has been shown that loss of miR-143 or miR-145 expression induces structural modifications of the aorta, impairing VSMC differentiation (Elia et al. 2009). In addition, we have recently studied miRNA133a with a pro-contractile function. In particular, the expression level of miR133a is inversely correlated with VSMC growth. In other words, its levels decrease after vascular injury, when VSMCs are primed to proliferate, and increase when VSMCs are coaxed back to quiescence. We have also shown that overexpression of miR-133a through Adeno-miR-133a reduces VSMC proliferation and migration both in vivo and in vitro. This is partly achieved through miR-133a-mediated suppression of Sp-1 and Moesin key components of the regulatory SRF network (Torella et al. 2011). TCM Vol. 23, No. 1, 2013

Table 1. Injury-regulated miRNAs with targets and biological effects within the vascular wall miRNAs

Regulation after injury

Biological effect

Molecular targets

miR-1 miR-21

k m

KLF4, PIM1 PTEN, PDCD4

miR-24 miR-26a miR-31 miR-133a miR-143 miR-145

k m m k k k

Pro-contractile Pro-synthetic or pro-contractile Pro-synthetic Pro-synthetic Pro-synthetic Pro-contractile Pro-contractile Pro-contractile

miR-146a miR-221 miR-222

m m m

Pro-synthetic Pro-synthetic Pro-synthetic

On the other hand, miR-1 increases the expression of contractile proteins through a repressive effect on KLF4 (Xie et al. 2011). In addition, myocardin induces miR-1 expression in VSMC. This is particularly interesting, since it demonstrates the involvement of different miRNAs, as miR-1 and the miR-143/145 cluster, in myocardin-dependent VSMC regulation, which is one of the most important regulatory pathways for VSMC phenotype modulation. In other words, different classes of regulatory molecules coexist and interact within the same signaling pathway (Chen et al. 2010). Although miR-21 is one of the earliest and most studied for cardiovascular disease, its role in the VSMC differentiation is still controversial. In fact, Ji et al. (2007) demonstrated that modulation of aberrantly overexpressed miR-21 significantly prevents neointimal lesion formation, since its depletion results in decreased cell proliferation and increased cell apoptosis, through modulation of PTEN and Bcl-2. In contrast, Davis et al. (2008) reported that miR-21 induces a suppression of programmed cell death 4 (PDCD4) levels, increasing the biosynthesis of contractile proteins in VSMC.

MiRNAs and Pro-synthetic Phenotype MiR-221 and miR-222 play an intriguing role as modulators of VSMC function and neointimal lesion formation. Both of them are significantly increased in proliferative VSMC, and their knockdown prevents VSMC proliferation and intimal thickening TCM Vol. 23, No. 1, 2013

TRB3 SMAD1/4 LATS2 SP-1, Moesin ELK1, FRA1 ACE, KLF4/5, CALMK, MRTFB KLF4 C-KIT, P27, P57 P27, P57

in rat carotid artery after vascular injury (Liu et al. 2012). Targets addressed by miR-221/222 in VSMC include p27 (Kip1), p57 (Kip2) and c-Kit—key regulators of VSMC proliferation (Liu et al. 2011). Looking at the net effect of miR221/222 on the vascular wall, differential effects are exerted on VSMC and EC. In fact, while they have pro-proliferative, promigratory and anti-apoptotic effects on VSMC, the same miRNAs can exert opposite effects on EC. The different expression profiles of their target genes, in the two cell types, are probably responsible for this discrepancy of effects. Interestingly, miR221/222 are up-regulated in circulating endothelial progenitor cells (EPCs) from patients with coronary artery disease (CAD) (Minami et al. 2009). Recently, an important regulatory role has been recognized to miR-146a in promoting VSMC proliferation in vitro and vascular neointimal hyperplasia in vivo (Sun et al. 2011). In fact, miR-146a is able to bind to ¨ the Kruppel-like factor 4 (KLF4) 30 untranslated region, with consequent regulation of its expression. In particular, silencing of miR-146a in VSMC increases KLF4 expression, whereas overexpression of miR-146a has an opposite effect (Sun et al. 2011). Similar to what observed in myocardin-dependent VSMC regulation, miRNAs are also involved in the PDGFBB signaling. In fact, miR-24 represses Tribble-like protein-3 (Trb3) mRNA, resulting in down-regulation of Smad proteins with an inhibitory effect on the TGF-b/ BMP-4 pathway. Consequently, inhibition of miR-24 function prevents PDGF-BBinduced switch of VSMC to a synthetic

phenotype (Chan et al. 2010). The regulatory function exerted by miR-31 on VSMC proliferation and neointimal formation represents a further example of interplay between different signaling pathways (Liu et al. 2011). Interestingly, inhibitors of mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) prevent up-regulation of miR-31, which in turn inhibits VMSC proliferation and neointimal formation (Liu et al. 2011). Also inhibition of miR-26a results in a structured response in VSMC, promoting cell differentiation, apoptosis and, at the same time, inhibiting proliferation and migration. These effects are achieved through modulation of TGFb signaling, since inhibition of miR-26a increases gene expression of SMAD-1 and SMAD-4 (Leeper et al. 2011).

MiRNAs and Endothelial Cells After Injury MiR-126 is highly expressed in EC (Harris et al. 2008). Its expression is regulated by several transcription factors, such as E26 transformation-specific sequences (ETSs) factor or KLF2 (Nicoli et al. 2010). Inhibition of miR-126 impairs vasculogenesis during early embryonic development and affects neo-vascularization after acute myocardial infarction, through modulation of VEGF signaling (Fish et al. 2008). In turn, miR-126 down-regulates VCAM1, reducing leukocyte adhesion on the endothelium (Harris et al. 2008), and up-regulates CXCL12, acting on its autoregulatory loop through RGS16-mediated activation of its receptor CXCR4. The net effect is plaque stabilization through reduced macrophage recruitment and increased EPC uptake (Zernecke et al. 2009). Cellular aging, and particularly EC aging, is an emerging issue in vascular biology due to its involvement in atherosclerosis. Through a microarray approach, Menghini et al. recently found miR-217 to be overexpressed in ‘‘aged’’ EC. This finding did not come as a surprise, as miR-217 regulates the expression of silent information regulator 1 (SirT1), a major regulator of longevity and metabolic disorders that is progressively reduced in multiple tissues during aging (Menghini et al. 2009). Table 2 summarizes the function of the most relevant miRs in EC. 11

Table 2. MicroRNAs implicated in endothelial cell function miRNAs

Molecular targets

Biological effect

miR-126

VCAM-1 KLF2/VEGFR-2 CXCL12

Cell adhesion and cell interactions Cell proliferation and migration Apoptosis and EPC recruitment

miR-217

SIRT1/FOXO

Vessel formation and maturation

TSP1/CTGF TSR/VEGFR-2 TSR/VEGFR-2 CYCLIN D1 VEGF ITG-a5

Cell proliferation and migration Cell proliferation and migration Cell proliferation and migration Cell proliferation and migration Cell adhesion and cell interactions

miR-221/222

c-kit eNOS

Cell proliferation and migration Vessel permeability

miR-663

FOSB/CEBPB

Inflammatory response

miR-17–92 miR-17-5p miR-18a miR-19a miR-20a miR-92a

Endothelial Cells Are Able to Sense Shear Stress Conditions Physiological shear stress regulates the expression of several atheroprotective genes, modulating the levels of KLF2 (Boon et al. 2009). On the contrary, reduced shear stress is responsible for switching EC toward a pro-atherogenic phenotype (Chatzizisis et al. 2007). Interestingly, EC response to shear stress is also mediated by miRNAs. In fact, miR10a was found to be down-regulated in EC from those regions of the aortic arch where physiological shear stress is altered, with consequent release of its inhibitory action on pro-inflammatory elements, such as nuclear factor (NF)-kB or mitogen-activated kinase kinase kinase 7 (MAP3K7) and b-transducin repeat-containing gene (TRC) (Fang et al. 2010). Although no direct evidence is available on the specific effects of shear stress on vascular remodeling after angioplasty, it is tempting to speculate that similar mechanisms mediate the response to the profound mechanical and biological alterations after balloon angioplasty or stenting. Any perturbation of tissue stretch can influence its biological homeostasis, with consequent effects on vascular remodeling (Lu et al. 2011). Additional examples include miR-19a, which is up-regulated by high shear stress and inhibits EC proliferation addressing cyclin D1 (Qin et al. 2010), or miR-663 which is instead induced under low shear stress and promotes 12

monocyte adhesion onto EC (Ni et al. 2011). Also the anti-angiogenic miR-17– 92 cluster is induced under low shear stress (Wu et al. 2011). In addition, since miR-92a targets KLF2, which is a transcriptional activator of miR-126, there is an interplay between miR-92a and miR-126, which is affected by shear flow conditions (Nicoli et al. 2010; Wu et al. 2011).

• Cell-to-Cell Crosstalk It has been recently demonstrated that miRNAs are also to be found outside the cells, in a surprisingly stable form. Interestingly, miRNAs can be actively exported or uptaken by cells, suggesting their involvement in cell-to-cell communication. Intercellular signaling has been reported between EC and VSMC through lipid vesicle-mediated transfer of RNAs (Zernecke et al. 2009). In addition, ECs have been shown to influence intracellular signaling of other cells through apoptotic bodies-mediated transfer of miR-126, which finally acts as a messenger signal on distant cells (Zernecke et al. 2009). These evidences apparently find a clinical correlate, as miR-126 has been shown to be selectively depleted upon passage through the coronary circulation (De Rosa et al. 2011). A recent study also supported this hypothesis, reporting that HUVECs secrete a specific set of miRNAs, which are shuttled by microvesicles (MVs), enabling

cell-to-cell trafficking of microRNAs from EC to VSMC (Hergenreider et al. 2012). Interestingly, the same mechanisms seem to mediate communication between cells of the vessel walls and different cell populations. For example, monocytes that release miR-150, which can be uptaken by EC, regulate their migration (Zhang et al. 2010).

• Future Experimental Directions MicroRNAs (miRNAs) represent a novel class of endogenous regulators of VSMC differentiation and phenotype switch, as well as of EC’s response to injury. Consistently, regulation of specific miRNAs during neointimal formation and reendothelialization after injury indicates dynamic regulation of miRNAs as a key determinant of the alternating state of VSMC. Consequently, the evidence that miRNAs are able to induce and maintain differentiation of VSMC raises the possibility that the manipulation of these miRNAs could prevent neointimal formation and foster re-endothelialization after injury. Since bare metal stent failure is mainly due to neointimal proliferation, an active area of investigations is exploring the possibility to prevent the restenosis using specific miRs, as miR133a or miR-145. Indeed, the number of candidate miRNAs to be exploited as a therapeutic target is increasing, as we are understanding more and more aspects of their function. Since we have recently observed that inhibition of miR-92a produces differential effects on EC and VSMC, reducing proliferation of VSMC and at the same time fostering re-endothelialization (Iaconetti et al. 2012), modulation of its expression levels may represent a novel strategy to improve endothelial regeneration and reduce restenosis after vascular injury. Therapeutic angiogenesis in critical limb ischemia or in myocardial infarction using specific microRNAs is also a promising area of research within the very last years (Thum, 2012). However, novel therapeutic strategies will face the major challenge of developing standardized methods for miRNAs inhibition, combining transfection efficiency and targeted delivery. Although methods are available to down-regulate miRNAs in vivo (local delivery of antimiRs via coated stents, coated balloon or TCM Vol. 23, No. 1, 2013

systemic administration of anti-miRs), miR mimics lack the favorable characteristics of anti-miRs strategies as they require more reliable and safe delivery systems. The hypothesis that miRNAs can act as endocrine molecules, transferring signaling between cells of the same or different tissue, is also very interesting and could be an interesting future research field.

• Clinical Perspectives Clinical failure of bare metal stents is due to neointimal proliferation, whereas stent thrombosis after drug-eluting stent (DES) implantation is mainly related to lack of endothelialization. Therefore, it is reasonable to explore the possibility to reduce VSMC proliferation using specific miRNAs, as miR-133a or miR-145. Finally, stent thrombosis could be specifically preventing manipulating specific miRNAs that increase endothelial regeneration after DES, as miR-92a or miR-126. Further clinical studies are needed to confirm in a clinical setting the promising data suggested by experimental studies.

• Funding The present work was partly supported by grants of the Italian Ministry of Health (Ricerca Finalizzata 2007, APICE Project), by GENECOR, a non-profit organization and by Boston Scientific. References Bartel DP: MicroRNAs: genomics, biogenesis, mechanism, and function. Cell 2004;116: 281–297. Boon RA, Horrevoets AJ, et al: Key transcriptional regulators of the vasoprotective effects of shear stress. Hamostaseologie 2009;29:39–40. Casscells W: Migration of smooth muscle and endothelial cells. Critical events in restenosis. Circulation 1992;86:723–729. Chan MC, Hilyard AC, Wu C, et al: Molecular basis for antagonism between PDGF and the TGFbeta family of signalling pathways by control of miR-24 expression. EMBO Journal 2010;29:559–573. Chatzizisis YS, Coskun AU, Jonas M, et al: Role of endothelial shear stress in the natural history of coronary atherosclerosis and vascular remodeling: molecular, cellular, and vascular behavior. Journal of the American College of Cardiology 2007;49:2379–2393. TCM Vol. 23, No. 1, 2013

Chen J, Yin H, Jiang Y, et al: Induction of microrna-1 by myocardin in smooth muscle cells inhibits cell proliferation. Arteriosclerosis, Thrombosis, and Vascular Biology 2010;31:368–375. Cheng Y, Liu X, Yang J, et al: MicroRNA-145, a novel smooth muscle cell phenotypic marker and modulator, controls vascular neointimal lesion formation. Circulation Research 2009;105:158–166. Cordes KR, Sheehy NT, White MP, et al: miR145 and miR-143 regulate smooth muscle cell fate and plasticity. Nature 2009; 460:705–710. Curcio A, Torella D, & Indolfi C: Mechanisms of smooth muscle cell proliferation and endothelial regeneration after vascular injury and stenting: approach to therapy. Circulation Journal 2011;75:1287–1296. Davis BN, Hilyard AC, Lagna G, et al: SMAD proteins control DROSHA-mediated microRNA maturation. Nature 2008;454:56–61.

smooth muscle cell proliferation after vascular injury in vivo. Nature Medicine 1995;1:541–545. Indolfi C, Avvedimento EV, Di Lorenzo E, et al: Activation of cAMP–PKA signaling in vivo inhibits smooth muscle cell proliferation induced by vascular injury. Nature Medicine 1997;3:775–779. Indolfi C, Di Lorenzo E, Rapacciuolo A, et al: 8-Chloro-cAMP inhibits smooth muscle cell proliferation in vitro and neointima formation induced by balloon injury in vivo. Journal of the American College of Cardiology 2000;36:288–293. Indolfi C, Torella D, Cavuto L, et al: Effects of balloon injury on neointimal hyperplasia in streptozotocin-induced diabetes and in hyperinsulinemic nondiabetic pancreatic islet-transplanted rats. Circulation 2001; 103:2980–2986.

De Rosa S, Fichtlscherer S, Lehmann R, et al: Transcoronary concentration gradients of circulating microRNAs. Circulation 2011; 124:1936–1944.

Indolfi C, Stabile E, Coppola C, et al: Membrane-bound protein kinase A inhibits smooth muscle cell proliferation in vitro and in vivo by amplifying cAMP-protein kinase A signals. Circulation Research 2001;88:319–324.

Elia L, Quintavalle M, Zhang J, et al: The knockout of miR-143 and -145 alters smooth muscle cell maintenance and vascular homeostasis in mice: correlates with human disease. Cell Death and Differentiation 2009;16:1590–1598.

Indolfi C, Torella D, Coppola C, et al: Physical training increases eNOS vascular expression and activity and reduces restenosis after balloon angioplasty or arterial stenting in rats. Circulation Research 2002;91(13): 1190–1197.

Fang Y, Shi C, Manduchi E, et al: MicroRNA10a regulation of proinflammatory phenotype in athero-susceptible endothelium in vivo and in vitro. Proceedings of the National Academy of Sciences of the United States of America 2010;107:13450–13455.

Indolfi C, Mongiardo A, Curcio A, et al: Molecular mechanisms of in-stent restenosis and approach to therapy with eluting stents. Trends in Cardiovascular Medicine 2003;13:142–148.

Fish JE, Santoro MM, Morton SU, et al: miR126 regulates angiogenic signaling and vascular integrity. Developmental Cell 2008;15: 272–284. Harris TA, Yamakuchi M, Ferlito M, et al: MicroRNA-126 regulates endothelial expression of vascular cell adhesion molecule 1. Proceedings of the National Academy of Sciences of the United States of America 2008;105:1516–1521. Hergenreider E, Heydt S, Tre´guer K, et al: Atheroprotective communication between endothelial cells and smooth muscle cells through miRNAs. Nature Cell Biology 2012;14:249–256. Iaconetti C, Polimeni A, Sorrentino S, et al: Inhibition of mir-92a increases endothelial proliferation and migration in vitro as well as reduces neointimal proliferation in vivo after vascular injury. Basic Research in Cardiology 2012;107:296–309. Indolfi C, Esposito G, Di Lorenzo E, et al: Smooth muscle cell proliferation is proportional to the degree of balloon injury in a rat model of angioplasty. Circulation 1995;92: 1230–1235. Indolfi C, Avvedimento EV, Rapacciuolo A, et al: Inhibition of cellular ras prevents

Indolfi C, Pavia M, Angelillo IF, et al: Drugeluting stents versus bare metal stents in percutaneous coronary interventions (a meta-analysis). American Journal of Cardiology 2005;95:1146–1152. Ji R, Cheng Y, Yue J, et al: MicroRNA expression signature and antisense-mediated depletion reveal an essential role of microRNA in vascular neointimal lesion formation. Circulation Research 2007;100:1579–1588. Leeper NJ, Raiesdana A, Kojima Y, et al: MicroRNA-26a is a novel regulator of vascular smooth muscle cell function. Journal of Cellular Physiology 2011;226:1035–1043. Liu X, Cheng Y, Chen X, et al: MicroRNA-31 regulated by the extracellular regulated kinase is involved in vascular smooth muscle cell growth via large tumor suppressor homolog 2. Journal of Biological Chemistry 2011;286:42371–42380. Liu X, Cheng Y, Yang J, et al: Cell-specific effects of miR-221/222 in vessels: molecular mechanism and therapeutic application. Journal of Molecular and Cellular Cardiology 2012;52:245–255. Lu D, Kassab GS, et al: Role of shear stress and stretch in vascular mechanobiology. Journal of the Royal Society Interface 2011;8:1379–1385. 13

Menghini R, Casagrande V, Cardellini M, et al: MicroRNA 217 modulates endothelial cell senescence via silent information regulator 1. Circulation 2009;120:1524–1532. Minami Y, Satoh M, Maesawa C, et al: Effect of atorvastatin on microRNA 221/222 expression in endothelial progenitor cells obtained from patients with coronary artery disease. European Journal of Clinical Investigation 2009;39:359–367. Ni CW, Qiu H, Jo H, et al: MicroRNA-663 upregulated by oscillatory shear stress plays a role in inflammatory response of endothelial cells. American Journal of Physiology: Heart and Circulatory Physiology 2011;300: 1762–1769. Nicoli S, Standley C, Walker P, et al: MicroRNA-mediated integration of haemodynamics and Vegf signalling during angiogenesis. Nature 2010;464:1196–1200. Qin X, Wang X, Wang Y, et al: MicroRNA-19a mediates the suppressive effect of laminar flow on cyclin D1 expression in human umbilical vein endothelial cells. Proceedings of the National Academy of Sciences of the United States of America 2010;107:3240–3244. Sun SG, Zheng B, Han M, et al: miR-146a and ¨ Kruppel-like factor 4 form a feedback loop to participate in vascular smooth muscle cell proliferation. EMBO Reports 2011;12:56–62. Thum T: MicroRNA therapeutics in cardiovascular medicine. EMBO Molecular Medicine 2012;4:3–14. Torella D, Iaconetti C, Catalucci D, et al: MicroRNA-133 controls vascular smooth muscle cell phenotypic switch in vitro and vascular remodeling in vivo. Circulation Research 2011;109:880–893. Wu W, Xiao H, Laguna-Fernandez A, et al: Flow-dependent regulation of Kruppel-like factor 2 is mediated by microrna-92a. Circulation 2011;124:633–641. Xie C, Huang H, Sun X, et al: MicroRNA-1 regulates smooth muscle cell differentiation by repressing Kruppel-like factor 4. Stem Cells and Development 2011;20:205–210. Zampetaki A, Willeit P, Drozdov I, et al: Profiling of circulating microRNAs: from single biomarkers to re-wired networks. Cardiovascular Research 2012;93:555–562. Zernecke A, Bidzhekov K, Noels H, et al: Delivery of microRNA-126 by apoptotic bodies induces CXCL12-dependent vascular protection. Science Signaling 2009;2:ra81. Zhang Y, Liu D, Chen X, et al: Secreted monocytic miR-150 enhances targeted endothelial cell migration. Molecular Cell 2010;39:133–144. PII: S1050-1738(12)00283-6

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Redox Regulation of Mitochondrial ATP Synthase Sheng-Bing Wang, Christopher I. Murray, Heaseung S. Chung, and Jennifer E. Van Eyk* Reversible cysteine oxidative post-translational modifications (Ox-PTMs) represent an important mechanism to regulate protein structure and function. In mitochondria, redox reactions can modulate components of the electron transport chain (ETC), the F1F0–ATP synthase complex, and other matrix proteins/enzymes. Emerging evidence has linked Ox-PTMs to mitochondrial dysfunction and heart failure, highlighting some potential therapeutic avenues. Ox-PTMs can modify a variety of amino acid residues, including cysteine, and have the potential to modulate the function of a large number of proteins. Among this group, there is a selected subset of amino acid residues that can function as redox switches. These unique sites are proposed to monitor the cell’s oxidative balance through their response to the various Ox-PTMs. In this review, the role of Ox-PTMs in the regulation of the F1F0–ATP synthase complex is discussed in the context of heart failure and its possible clinical treatment. (Trends Cardiovasc Med 23:14-18) C 2013 Elsevier Inc. All rights reserved.

• Introduction Reactive oxygen/nitrogen species (ROS/ RNS) have a dual nature in the cell. Excess generation of ROS/RNS contributes to the development and progression of many

Sheng-Bing Wang is at the Division of Cardiology, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD 21224, USA. Christopher I. Murray was at the Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21224, USA and he is currently at University of British Columbia, Vancouver, BC, Canada V6T 124. Heaseung S. Chung is at the Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21224, USA. Jennifer E. Van Eyk is at the Division of Cardiology, Department of Medicine and Department of Biological Chemistry, School of Medicine, Johns Hopkins University, Baltimore, MD 21224, USA. *Address correspondence to: Division of Cardiology, Department of Medicine, School of Medicine, Johns Hopkins University, Baltimore, MD 21224, USA. Tel.: þ1 410 550 8510; fax: þ1 410 550 8512; e-mail: [email protected]. & 2013 Elsevier Inc. All rights reserved. 1050-1738/$-see front matter

diseases, including cardiovascular disease; however, increasing evidence suggests that lower concentrations of ROS/RNS can contribute to cellular signaling (Valko et al. 2007). The mitochondria produce the majority of cellular ATP via the concerted actions of the electron transport chain (ETC) and the F1F0–ATP synthase (Boyer 1997). The ETC is also a primary source of ROS/RNS production in the mitochondria, which has been found to impact the F1F0– ATP synthase complex. There is considerable knowledge about the structure and function of F1F0–ATP synthase and its subunits (Ackerman and Tzagoloff 2005; Feniouk and Yoshida 2008; Pedersen 2007). In the last few years, a number of phosphorylated amino acid residues have been identified (Agnetti et al. 2010; Arrell et al. 2006; Deng et al. 2011) and, in some cases, shown to alter the function of the F1F0–ATP synthase complex (Kane et al. 2010). Until recently, little was known regarding the Ox-PTM-dependent regulation of ATP synthase subunits. Advances in PTM-specific proteomic methods (antibody and mass spectrometry based) for OxPTMs of cysteine residues have allowed for the detection of a surprising number of TCM Vol. 23, No. 1, 2013