Vascular smooth muscle cell proliferation is dependent upon upregulation of mitochondrial transcription factor A (mtTFA) expression in injured rat carotid artery

Atherosclerosis 178 (2005) 39–47

Vascular smooth muscle cell proliferation is dependent upon upregulation of mitochondrial transcription factor A (mtTFA) expression in injured rat carotid artery Tomonori Yoshida, Hiroyuki Azuma∗ , Ken-ichi Aihara, Mitsunori Fujimura, Masashi Akaike, Takao Mitsui, Toshio Matsumoto Department of Medicine and Bioregulatory Sciences, University of Tokushima Graduate School of Medicine, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan Received 16 February 2004; received in revised form 17 July 2004; accepted 10 August 2004 Available online 7 October 2004

Abstract Consistent with the physiological response to increased energy demand in proliferating cells, the number of mitochondria is upregulated in synthetic states of vascular smooth muscle cells (VSMC) in atherosclerotic lesion. We hypothesized that mitochondrial transcription factor A (mtTFA), a prerequisite factor for the transcription and replication of mtDNA, may be upregulated in VSMC of injured rat carotid artery, and that inhibition of its expression can attenuate the intimal thickening. Changes of intimal thickening and mtTFA expression by a treatment with antisense oligodeoxynucleotides (ODN) for mtTFA were investigated in balloon-injured rat carotid artery model. The expression of mtTFA was upregulated as early as 3 h up to 7 days after balloon injury. Delivery of ansisense ODN for mtTFA from adventitia side to injured arterial wall caused a significant decrease in intima-to-media (I/M) ratio. Furthermore, the increase in immunoreactivity and mRNA expression of mtTFA in injured artery as well as the number of mitochondria in intimal VSMC was abrogated by antisense ODN treatment. These data demonstrate that expression of mtTFA is upregulated in intimal VSMC of injured rat carotid artery, and that suppression of mtTFA expression by antisense ODN can attenuate intimal thickening after balloon injury. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Antisense oligodeoxynucleotide; Arterial injury; Intimal thickening; Dedifferentiation; Restenosis

1. Introduction Recent advances in revascularization technologies provided long-term disease-free periods to many patients with coronary vascular diseases. In particular, stent implantation has become a major therapeutic procedure with excellent immediate outcome. Despite these successful outcomes, 7–37% of patients with coronary stent implantation are reported to develop angiographic restenoses and require additional treatment [1]. Histopathological studies of these restenotic lesions revealed that restenoses after plain old balloon angioplasty ∗

Corresponding author. Tel.: +81 88 633 7120; fax: +81 88 633 7121. E-mail address: [email protected] (H. Azuma).

0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.08.029

or stenting are distinct from primary atherosclerotic lesions [2,3]. Atherosclerotic lesions consist of such findings as infiltration of macrophages and lymphocytes, proliferation of smooth muscle cells, intracellular and interstitial lipid accumulation, and fibrin deposition. In contrast, restenotic lesions are principally composed of intimal hyperplasia and vascular negative remodeling. Although stenting prevents negative remodeling, stent in itself worsens intimal hyperplasia. In addition, recent studies with drug-eluting stents have shown excellent outcomes in reducing the restenosis rate of de novo lesions to <5% [4]. In particular, clinical trials with sirolimus (rapamycin)-eluting stents showed longterm inhibition of neointimal hyperplasia for up to 2 years [5].

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Restenosis caused by intimal hyperplasia is mainly a consequence of dedifferentiation, migration and proliferation of vascular smooth muscle cells (VSMC) [6]. Although many growth factors and transcriptional factors such as platelet derived growth factor (PDGF), transforming growth factor (TGF)-␤, NF-␬ B, Egr-1 and AP-1 have been postulated to be candidate molecules for phenotypic modulation from contractile state to synthetic state of VSMC [7,8], a key molecule that regulates these processes remains elusive. Electron microscopic analyses demonstrated that synthetic state of VSMC contains a large number of cellular organelles such as ribosomes, rough endoplasmic reticulum, Golgi complexes, and mitochondria in its cytoplasm [9]. In particular, an enhancement of cell growth and division is accompanied by an increase in mitochondria, because under such circumstances cells, need to generate high energy through oxidative phosphorylation. Thus, mammalian mitochondria must enhance the transcription and replication of mitochondrial DNA (mtDNA) to respond to the increased cellular ATP demands and mitogenesis. Transcription and replication of mtDNA require mitochondrial RNA polymerase, mitochondrial transcriptional factor A (mtTFA), mitochondrial termination factor of transcription (mTERF) [10], and another newly identified mitochondrial transcriptional factor either TFB1M or TFB2M [11]. Among these mitochondrial factors, mtTFA has been most extensively studied, and appears to play an important role in mitochondrial gene replication under increased ATP demands. Thus, deficiency of mtTFA is known to elicit myopathy in humans [12], and mtTFA gene knockout causes both cardiomyopathy and myopathy in mice [13]. Those observations are consistent with the hypothesis that adaptive mitochondrial biogenesis in metabolically active cells can be achieved through an enhancement of the transcription and replication of mtDNA by an upregulation of mtTFA. If this is the case, the expression of mtTFA is expected to be enhanced in intimal VSMC of injured artery, and intimal hyperplasia observed after arterial injury can be suppressed by abrogating the adaptive enhancement of mtTFA expression. To test this hypothesis, we investigated changes in the expression of mtTFA protein and mRNA, and effects of antisense oligodeoxynucleotides (ODN) for mtTFA on intima-to-media (I/M) ratio using balloon-injured rat carotid artery model.

2. Material and methods 2.1. Balloon injury Male Sprague–Dawley (SD) rats weighing 350–480 g were obtained from Japan SLC Inc. (Hamamatsu, Japan). All animals were provided with care guided by National Institutes of Health (Guide for the Care and Use of Laboratory Animals, NIH publication No. 86-23, revised 1985). Rats were anesthetized with sodium pentobarbital intraperi-

toneally (30 mg/kg). Left common carotid arteries were exposed through a midline neck incision. A 2-french Fogarty balloon catheter was introduced through an arteriotomy to the right common femoral artery, advanced into the common carotid, inflated to generate resistance and withdrawn three times. After removal of the catheter, a ligature was applied to the right common femoral artery proximal to the arteriotomy. The incision was closed and the rats were allowed to recover. Rats were killed by lethal injection of phenobarbitone, and arteries were harvested at 3 h, 24 h, 7 days and 14 days after the procedure. 2.2. Immunohistochemistry Expression and distribution of mtTFA protein in ballooninjured carotid artery was detected by immunohistochemical analysis. Cryostat arterial sections were fixed with 4% paraformaldehyde in buffer A (DPBS containing 0.05% Tween 20) at room temperature for 15 min, washed with buffer A twice, permeabilized with 0.5% TritonX-100 in buffer A for 5 min, and washed again three times with buffer A. Sections were blocked in buffer B (3% BSA and 5% normal rabbit serum in buffer A) for 20 min and reacted with a rabbit polyclonal anti-human mtTFA antibody (1:1000 dilution in buffer B), which was kindly provided by Dr. Clayton, for 1 h at room temperature. After washing three times with buffer A, the sections were incubated with biotinylated goat anti-rabbit IgG (DAKO JAPAN, Kyoto, Japan, 1:400 dilution in buffer B) for 1 h, washed, and immunostained using a DAKO ENVISION kit/HRP (DAB). 2.3. Preparation and treatment procedure of antisense oligodeoxynucleotides (ODN) We prepared one sense, one scramble and three different kinds of antisense ODN. ODN were phosphorothioatesubstituded in all positions and HPLC-purified (Sysmex Corp., Kobe, Japan). The sequences of sense (S) and scramble (SCR) ODN were: 5 -GGTGTATGAAGCGGATTTTAA-3 , corresponding to nucleotide numbers 105–125 (numbering according to Mezzina et al. [14]) and 5 -GATCGATCGATCGATCGATCG-3 , respectively. The three kinds of antisense ODN, A1, A2, and A3 were 5 -CTTCATACACCTTTTTTTCTG-3 , corresponding to 95–115, 5 -ATCCGCTTCATACACCTTTTT-3 , corresponding to 99–120, and 5 -TTAAAATCCGCTTCATACACC-3 , corresponding to 105–125, respectively. ODN were suspended in Tris–EDTA (10 mmol/L Tris, pH 7.4, and 1 mmol/L EDTA, pH 8.0), mixed with 25% (w/v) of a nonionic, surfactant polyol Pluronic® F-127 gel (Pluronic gel, Molecular Probes Inc., Eugene, OR) at a concentration of 0.4 mmol/L, and stored at 4 ◦ C. Pluronic gel containing 1.04 mmol/L of each ODN or Tris–EDTA (vehicle, V) was pasted around the injured left common carotid artery immediately after endothelial denudation.

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2.4. Fluorescence microscopy FITC-labeled A1 ODN (1 ␮mol/L) or Tris–EDTA in Pluronic gel was pasted around the injured carotid artery as described above. At 6 h after the procedure, arteries were harvested, immediately embedded in OCT compound (Tissue Tek® , Miles Inc., Elkhart, IN), and frozen in dry ice/acetone. Four-micron serial sections were cut and analyzed using Nikon ECLIPSE E600 fluorescence microscope (Tokyo, Japan). 2.5. Morphometric analysis Effects of ODN on intimal thickening were evaluated at 14 days after injury. Carotid arteries were perfusionfixed using 10% formaldehyde (v/v) at 120 mm Hg, dissected from rats, and placed in 10% formaldehyde. Injured segments were excised along with proximal and distal uninjured segments and further fixed in 10% formaldehyde overnight. Fixed vessels were cut into 2-mm segments, dehydrated, and embedded in paraffin. One section from each of five segments was stained with Elastica-van Gieson and hematoxylin-eosin. Morphometric analysis of arterial sections was performed with a computerized digital image analysis system (the public domain NIH-image program) in a blind manner. Areas within the external elastica lamina (EEL area) and the internal elastic lamina (IEL area) and the lumen area were measured, thereby I/M ratio was calculated by fol-

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lowing formula: (IEL area − lumen area)/(EEL area − IEL area). 2.6. Reverse transcription-polymerase chain reaction (RT-PCR) analysis of mtTFA mRNA mRNA levels of mtTFA were assessed by a semiquantitative RT-PCR technique at 3 h, 24 h, 7 days and 14 days after injury. Arterial segments were dissected out, immediately placed in 4 mol/L guanidine isothiocyanate and homogenized. Total RNA was isolated using TRIZOL® Reagent (Invitrogen, San Diego, CA), quantitated by absorbance at 260 nm, and 1 ␮g of RNA in 20 ␮L of volume was reverse transcribed with oligo (dT) primer using TM AccesseQuick RT-PCR System (Promega, Madison, WI). One microliter of the reverse transcription reaction mixture was then subjected to PCR for Taq polymerase (Takara Shuzo Co., Shiga, Japan) using both the forward primer 5 -GAGCAGCTAACTCCAAGTCAG-3 , corresponding to 169–189 (numbering according to Mezzina et al. [14]), and the reverse primer 5 -ATTCTATCATCTTTAGCAAGC3 corresponding to 420–440. The samples were amplified for 28 cycles using the following denaturation, annealing, and extension conditions: 95 ◦ C for 1 min, 55 ◦ C for 1 min, and 68 ◦ C for 1 min, respectively. The amplified products were confirmed to be a rat mtTFA cDNA by sequencing analysis. Glyceraldehyde-3-phosphate dehydrogenase (G3PDH) mRNA was used as a control.

Fig. 1. Immunohistochemical analysis of mtTFA in rat carotid artery. (A and D) Uninjured carotid artery. (B and E) Injured carotid artery at 7 days after balloon injury. (C and F) Injured carotid artery at 14 days after balloon injury. Immunoreactivity of mtTFA was detected in intimal VSMC at both 7 days and 14 days after balloon injury and its expression level was more intensive at 7 days than at 14 days. Arrow indicates IEL. Bars = 50 ␮m.

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2.7. Semi-quantitative PCR analysis of mtDNA contents Mitochondrial DNA levels in injured arteries were assessed by a semi-quantitative PCR technique at 7 days after injury. Arterial segments were dissected out, and genomic DNA was immediately extracted using Wizard® Genomic DNA Purification Kit (Promega). DNA samples were quantitated by absorbance at 260 nm, and 10 ng of DNA in a 20 ␮L of volume was subjected to PCR for Taq polymerase (Takara Shuzo Co.) using both the forward primer 5 GCAGTATTCGCCATCATAGCTGG-3 , corresponding to 6461–6483 (numbering according to Gadaleta et al. [15]), and the reverse primer 5 -GCCTATAGAGGAGACTGTATTTC3 , corresponding to 6657–6679, of rat mtDNA sequence. The samples were amplified for 25 cycles using the following denaturation, annealing, and extension conditions: 94 ◦ C for 1 min, 55 ◦ C for 1 min, and 72 ◦ C for 1.5 min, respectively. The amplified products were confirmed to be a rat mtDNA by sequencing analysis. G3PDH DNA was used as a control. 2.8. Preparation for electron microscopic analysis Arterial segments were fixed with 3% glutaraldehyde solution in 0.1 mol/L sodium cacodylate–HCl buffer (pH 7.3) containing 0.05 mol/L sucrose. For electron microscopy, the specimens were further fixed for 2 h at 4 ◦ C with 2% osmium tetroxide in 0.1 mol/L cacodylate buffer (pH 7.3) containing 0.5% potassium ferrocyanate, dehydrated in graded ethanol (70–100%), stained with 2% uranyl acetate in ethanol, and embedded in Spurr low viscosity epoxy resin. The sections were cut with a diamond knife on an LKB Ultrotome IV, picked up on carbon-coated Formvar films, stained with alkaline lead citrate, and examined by a JOEL 100CX electron microscope at 60 kV (Kodak MN film 4489, Eastman Kodak Company, Rochester, NY).

Fig. 2. Time course of mRNA expression of mtTFA in injured rat carotid artery. (A) Representative semi-quantitative RT-PCR analysis of mtTFA mRNA in rat carotid artery at 0 h, 3 h, 24 h, 7 days and 14 days after injury from three experiments. MW represents DNA molecular size marker. (B) Quantitative analysis of the intensity of bands in (A). mRNA level of mtTFA was reached maximum at 24 h after balloon injury. Values show mean ± S.E.M. (n = 3).

Fig. 3. Immunofluorescence analysis of FITC-labeled ODN. FITC-labled A1 ODN (1 ␮mol/L) was mixed with Pluronic gel and pasted around the injured artery. Detection of FITC signal was performed at 6 h after balloon injury. Widespread FITC signal was detected throughout the injured arterial wall (B), but only background signal was observed when vehicle-containing Pluronic gel was pasted (A). Original magnification ×200.

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2.9. Statistical analysis All values were expressed as mean ± S.E.M. The data were statistically analyzed using one-way ANOVA followed by unpaired Student’s t-test with Fisher’s protected least significant difference for multiple comparisons. Statistical significance was determined by P < 0.05.

3. Results 3.1. Enhanced immunoreactivity of mtTFA in intimal VSMC after balloon injury In uninjured carotid artery, immunoreactivity of mtTFA was observed only in endothelial cells but not in VSMC of medial layer (Fig. 1A and D). At 7 days after balloon injury, immunoreactivity of mtTFA was intensely detected in VSMC of whole layers of intima (Fig. 1B and E). When carotid artery at 14 days after injury was examined, immunoreactivity of mtTFA was still observed in VSMC of intima. However, the staining of mtTFA in intimal VSMC at 14 days after balloon injury became less than that at 7 days, and only one or two layers from luminal surface of intimal VSMC were positive for mtTFA (Fig. 1C and F).

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3.2. Enhanced mRNA expression of mtTFA in injured artery Expression of mtTFA mRNA in injured artery at 3 h, 24 h, 7 days and 14 days after balloon injury was examined by a semi-quantitative RT-PCR analysis. As shown in Fig. 2, mRNA levels encoding mtTFA were upregulated as early as 3 h after injury and reached the maximum value after 24 h (approximately 1.4-fold of basal level). The mRNA levels remained elevated at 7 days after injury, declined thereafter and reached the baseline by 14 days. 3.3. Distribution of ODN in injured arterial wall To examine whether ODN pasted around the injured artery can infiltrate arterial wall to reach luminal surface, FITClabeled A1 ODN was prepared, mixed with Pluronic gel and pasted around the injured artery. As shown in Fig. 3B, widespread FITC signals throughout the injured arterial wall were observed at 6 h after the procedure, while only background fluorescence was detected in injured artery pasted vehicle-containing Pluronic gel (Fig. 3A). Thus, our strategy to paste ODN around the injured artery is demonstrated to be a valid approach to examine the effects of antisense ODN on intimal thickening after balloon injury.

Fig. 4. Effects of ODN treatment on intimal thickening after balloon injury. Representative light microscopic findings of rat carotid artery stained with Elasticavan Gieson at 14 days after balloon injury are shown. Vehicle (V), scramble ODN (SCR), sense ODN (S), or one of three kinds of antisense ODN (A1, A2 or A3) containing Pluronic gel was pasted around the injured artery and analyzed those effects on intimal thickening. Bars = 50 ␮m.

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Table 1 Intima-to-media (I/M) ratio of rat carotid artery after balloon injury with or without ODN treatment Group

Number

I/M ratio

P-values vs. vehicle

Vehicle Scramble Sense

6 4 4

0.951 ± 0.049 0.984 ± 0.042 1.021 ± 0.054

– NS NS

Antisense A14 A2 A3

4 4 4

0.557 ± 0.047 0.826 ± 0.057 0.707 ± 0.049

P < 0.05 NS P < 0.05

Intimal (I) and medial (M) areas were measured and I/M ratio was calculated as described in Section 2. Data are means ± S.E.M. of the indicated number of experiments. NS: not significant.

3.4. Effects of antisense ODN for mtTFA on intimal thickening after balloon injury Effects of five kinds of ODN, one scramble, one sense and three antisense ODN for mtTFA, on the degree of intimal thickening were evaluated morphologically using I/M ratio at 14 days after balloon injury. Fig. 4 shows representative morphological changes of intimal thickening by ODN treatment. As shown in Table 1, both A1 and A3 ODN significantly reduced I/M ratio as compared with the vehicle treatment, and the effect of A1 ODN was stronger than that of A3 ODN. A2 ODN reduced I/M ratio only slightly with no statistical significance. No inhibitory effect was observed in scramble and sense ODN-treated arteries. 3.5. Effects of antisense ODN on mtTFA mRNA expression after balloon injury To analyze the effects of antisense ODN on mtTFA mRNA expression, a semi-quantitative RT-PCR was performed using injured arteries. When sense ODN-treated arteries were examined, mRNA levels of mtTFA were unaffected as compared to vehicle-treated arteries (Fig. 5). When the effects of antisense ODN were examined, all of A1, A2 and A3 ODN significantly (P < 0.05) reduced mRNA levels of mtTFA, with the greatest efficacy in A1 ODN followed by A3 ODN and A2 ODN. Thus, the degree of suppression of mtTFA mRNA expression by antisense ODN was in parallel with the reduction of I/M ratio. 3.6. Effects of antisense ODN on mtTFA immunoreactivity after balloon injury We then performed immunohistochemical analysis to assess the effects of antisense ODN treatment on changes of mtTFA protein expression in arteries at 7 days after balloon injury. Immunoreactivity of mtTFA in vehicle-treated artery was intensely detected in VSMC of whole layers of intima (Fig. 6A–C), while the staining of mtTFA in A1 ODN-treated arteries was reduced compared to that in vehicle-treated arteries (Fig. 6D–F).

Fig. 5. Effects of ODN treatment on mRNA expression of mtTFA in injured artery. (A) A semi-quantitative RT-PCR analysis of mtTFA mRNA was performed using injured artery treated with or without ODN. PCR products were electrophoresed in 2% agarose gel. Data show a representative result from three experiments. V: vehicle; S: sense; A1: antisense 1; A2: antisense 2 and A3: antisense 3. MW shows DNA molecular size marker. (B) Quantitative analysis of the intensity of bands in (A). Each bar represents mean ± S.E.M. * P < 0.05.

3.7. Effects of antisense ODN on mtDNA expression after balloon injury To analyze the effects of antisense ODN on mitochondrial proliferation, electron microscopic analysis and semiquantitative PCR for mtDNA expression were performed using injured arteries. When vehicle or sense ODN-treated arteries were examined, both the number of mitochondria in intimal VSMC and mtDNA expression in injured artery were unaffected (Fig. 7). In contrast, the number of mitochondria in intimal VSMC as well as the expression levels of mtDNA in A1 ODN-treated arteries were reduced compared to vehicleand sense ODN-treated arteries (Fig. 7).

4. Discussion In this study, we found for the first time that expression of mtTFA is upregulated in dedifferentiated intimal VSMC of rat carotid artery after balloon injury, and that suppression of

T. Yoshida et al. / Atherosclerosis 178 (2005) 39–47

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Fig. 6. Effects of ODN treatment on mtTFA immunoreactivity in injured artery. Injured carotid artery at 7 days after balloon injury was immunohistochemically analyzed using mtTFA polyclonal antibody. (A–C) Artery treated with vehicle. (D–F) Artery treated with antisense ODN (A1). Immunoreactivity of mtTFA in A1 was reduced compared to that in vehicle. Arrow indicates IEL. Bars = 50 ␮m.

mtTFA mRNA expression by antisense ODN can attenuate the severity of intimal thickening after arterial injury. Phenotypic modulation of VSMC from contractile to synthetic states is a major cause of restenosis after percutaneous coronary intervention (PCI). VSMC at a contractile state is largely filled with well-organized myofilaments, dense bodies and dense membranes, whereas synthetic VSMC contains a large amount of cellular organelles such as ribosome, rough endoplasmic reticulum, Golgi complexes and mitochondria [16]. Many regulators of VSMC proliferation have been reported, including mechanical stress, growth factors, signal transduction molecules and transcription factors [17], and mitochondrial biogenesis must be enhanced under the stimulation of these regulators for VSMC proliferation. It is reported that approximately 1000 genes participate in mitochondrial biogenesis, and that more than 95% of these are encoded by the nucleus [18]. In particular, replication and transcription of mtDNA is the most crucial process in mitochondrial biogenesis, and requires nuclear genes such as mitochondrial RNA polymerase, mTERF, TFB1M or TFB2M, and mtTFA [19]. Among these factors, Larsson et al. demonstrated that mtTFA regulates mtDNA copy number and is essential for mitochondrial biogenesis, thereby leading to a respiratory chain dysfunction in heterozygous mtTFA knockout mice [20]. They also showed that the sensitivity to a reduction of mtTFA expression is different by organs, and that the heart is more sensitive than kidney or skeletal muscle using heart-specific disruption of mtTFA [21]. These observations further confirmed the importance of mtTFA in heart

development. In addition, reduction of mtTFA expression in cell culture study using antisense technique was associated with a suppression of mtDNA-encoded protein expression including cytochrome c oxidase subunit I and III [22]. In addition, over expression of mtTFA in cultured cells resulted in an increase in cytochrome c oxidase subunit I [23]. Taken together, it is plausible to assume mtTFA as a novel target for inhibiting VSMC proliferation in patients with restenosis after PCI. As a high mobility group protein with molecular weight of 25 kDa, mtTFA is shown to wrap, bend and unwind DNA by binding to upstream of the light- and heavy-strand mtDNA promoters, thereby accelerating transcription and replication of mtDNA [19]. Among clinical disorders, mtTFA expression is known to be enhanced in hyperthyroidism [24]. In rats, chronic electrical contractile stimulation is reported to enhance mtTFA expression in skeletal muscle [25]. In the present study, we identified that the expression of mtTFA is upregulated almost exclusively in proliferating intimal VSMC in rat carotid artery injury model. Although molecular mechanisms causing the upregulation of mtTFA expression in intimal VSMC after vascular injury is as yet unclear, promoter assay of mtTFA gene in cultured VSMC is in progress to identify major regulators involved in this process. Restenosis rate at 6 months after PCI is 20–50% in patients who underwent balloon angioplasty, and 10–30% in patients treated with stent implantation [26,27]. To prevent restenosis after PCI, systemic pharmacological intervention in combination with drug-eluting coronary stent implantation

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Fig. 7. Effects of ODN treatment on mitochondrial proliferation in injured artery. (A) Electron microscopic analysis of intimal VSMC at 7 days after balloon injury. The number of mitochondria in cytoplasm of VSMC was reduced in antisense ODN (A1) treatment compared in vehicle (V) or sense ODN (S) treatment. Bars = 50 ␮m. (B) PCR analysis of mtDNA contents in intimal VSMC. DNA isolated from artery at 7 days after balloon injury was amplified using primers for mtDNA or G3PDH and electrophoresed in 2% agarose gel. The expression levels of mtDNA in A1 ODN-treated arteries were reduced compared to vehicleand sense ODN-treated arteries. MW shows DNA molecular size marker. (C) Quantitative analysis of the intensity of bands in (B). Each bar represents mean ± S.E.M. ∗ P < 0.05.

is reported to be effective [27]. In addition, vascular gene therapies using antisense ODN, decoy ODN, ribozymes and DNAzymes are shown to reduce the incidence of restenosis by inhibiting VSMC growth [28]. Among these, antisense ODN strategy is most extensively investigated and some antisense ODN are now under clinical trials as therapeutic tools for the management of restenosis after PCI [28]. Finally, an important issue for the efficacy of antisense ODN strategy is the delivery systems. We employed a straightforward system that passively delivers ODN from adventitia to intimal side with successful attenuation of intimal thickening in rat carotid artery injured model. However, to deliver ODN more effectively and safely, alternative approaches such as stent-based delivery system may be needed for clinical application.

Conflict of interest Author(s) hereby acknowledge the AHA’s conflict of interest policy requirement to scrupulously avoid direct and

indirect conflicts of interest and, accordingly, hereby agrees to promptly inform the editor or editor’s designee of any business, commercial or other proprietary support, relationships or interests that the author(s) may have, which relate directly or indirectly to the subject of the work. Acknowledgments We would like to thank Dr. David Clayton of the Stanford University School of Medicine for providing rabbit antihuman mtTFA polyclonal antibody. This work was supported in part by a Grant-in-Aid for Priority Areas from the Ministry of Education, Science, Sports, and Culture of Japan. References [1] Lowe HC, Oesterle SN, Khachigian LM. Coronary in-stent restenosis: current status and future strategies. J Am Coll Cardiol 2002;39:183–93.

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