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Cardiovascular Research 2003 57(1):265-276; doi:10.1016/S0008-6363(02)00607-7
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Copyright © 2003, European Society of Cardiology

Chimeric DNA–RNA hammerhead ribozyme targeting PDGF A-chain mRNA specifically inhibits neointima formation in rat carotid artery after balloon injury

Motoko Kotani, Noboru Fukuda*, Hideyuki Ando, Wen-Yang Hu, Satoshi Kunimoto, Satoshi Saito and Katsuo Kanmatsuse

Second Department of Internal Medicine, Nihon University School of Medicine, Tokyo 173-8610, Japan

nhukuda{at}med.nihon-u.ac.jp

* Corresponding author. Tel.: +81-3-3972-8111; fax: +81-3-3972-1098

Received 5 April 2002; accepted 16 July 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Restenosis of the coronary artery after percutaneous transluminal coronary angioplasty (PTCA) occurs in 30–50% of patients and remains a major clinical problem. We developed ribozyme that targets platelet-derived growth factor (PDGF) A-chain mRNA as a gene therapy for restenosis after PTCA. Thus, we examined the effects of a chimeric DNA–RNA ribozyme targeting PDGF A-chain mRNA on neointima formation in rat carotid artery after balloon injury and evaluated its specificity for PDGF A-chain mRNA by microarray analysis. Methods: Rat carotid artery was injured with a 2F Fogarty catheter, and PDGF A-chain specific ribozyme was delivered to the injured artery with polyethylenimine. Two weeks after injury, the artery was removed, and the intima/media (I/M) ratio was evaluated. Six hours after injury, mRNA was extracted with oligo dT cellulose, and expression of PDGF A-chain mRNA was evaluated by reverse transcription-polymerase chain reaction. Expression of PDGF-AA protein was evaluated by Western blot analysis. Expression of 970 genes was evaluated by microarray (GeneChip, Affimetrix Inc). Results: FITC-labeled ribozyme was taken up into the midlayer smooth muscle of the carotid artery until 24 h after balloon injury. Two and 5 µg of ribozyme significantly reduced neointima formation by 44 and 55% of control levels, respectively, in a dose-dependent manner. Ribozyme markedly inhibited expression of PDGF A-chain mRNA as well as production of PDGF-AA protein in injured vessels. Microarray analysis revealed that expression of 525 genes was increased after balloon injury. These genes included FLK-1, interleukin-1 receptor, retinoic acid receptor {alpha}2 isoform, heat shock protein, MAP kinase kinase, Fas antigen, G6Pase, PI-5-P-kinase, p38 MAP kinase, proliferating cell nuclear antigen, transforming growth factor-β, extracellular signal-related kinase, and fibroblast growth factor receptor. With respect to expression of cytokine and growth factor mRNAs, the best ribozyme specifically inhibited expression of PDGF A-chain mRNA. Conclusions: Our chimeric DNA–RNA hammerhead ribozyme targeting PDGF A-chain mRNA inhibited neointima formation in rat carotid artery after balloon injury with specific inhibition of expression of PDGF A-chain mRNA, suggesting that this ribozyme may be useful for therapy of restenosis of coronary artery after PTCA.

KEYWORDS Angioplasty; Gene expression; Gene therapy; Growth factors; Restenosis


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Restenosis of coronary artery after percutaneous transluminal coronary angioplasty (PTCA) occurs in 20–30% of patients [1,2], and it remains a major clinical problem. Neointima formation with vascular smooth muscle cell (VSMC) hyperplasia is believed to play a critical role in restenosis [3]. Despite intensive trials, no effective therapy to prevent restenosis has been identified. Therefore, gene therapies, including molecular strategies, such as antisense oligodeoxynucleotides (ODN), ribozymes, and decoys are being assessed for treatment of restenosis.

Neointimal VSMCs have been reported to be changed to the synthetic phenotype compared to medial VSMCs [4] and produce several growth factors including basic fibroblast growth factor (bFGF), platelet-derived growth factor (PDGF) A-chain, epidermal growth factor [5], and transforming growth factor-β [6]. PDGF is a dimer composed of disulfide-linked A-chain and B-chain [7]. Nilsson et al. [8] showed that normal, growth-arrested VSMCs do not express PDGF mRNA, whereas cultured VSMCs or VSMCs in atherosclerotic plaques express PDGF A-chain mRNA and secrete PDGF-AA protein, indicating that PDGF A-chain contributes to VSMC proliferation in arterial proliferative disease.

Engineered inactivation of gene function is important for elucidating the function of a particular gene and may also serve as a gene therapy for treatment of viral infection, cancer, other diseases caused by aberrant gene expression. Gene function can be inactivated by antisense ODN at the DNA level or by ribozymes at the RNA level. Ribozymes hybridize and cleave target RNAs. Once the target has been cleaved, the ribozyme can dissociate from the cleaved transcript and repeat the process with another RNA molecule [9]. The major advantage of ribozymes is that they can sequence-specifically cleave multiple target mRNA molecules, whereas antisense molecules do not cleave the target molecules and act only at an equimolar ratio [10,11]. We designed and synthesized a chimeric DNA–RNA hammerhead ribozyme that targets the PDGF A-chain and examined its effects on the exaggerated growth of VSMCs from spontaneously hypertensive rats (SHR). We found that the ribozyme effectively and specifically inhibited the exaggerated growth of VSMCs from SHR in vitro. This effect was mediated the cleavage of the rat PDGF A-chain mRNA and resulting reduction of rat PDGF-AA protein production [12].

Methods for high-throughput analyses of gene expression have been described. These methods are based on microarrays of oligonucleotides that are used as gene-specific hybridization targets to quantitatively expression of the corresponding genes [13]. With these technologies, expression of hundreds to thousands of genes can be examined simultaneously [14].

In the present study, we examined the effect of a chimeric DNA–RNA ribozyme targeting PDGF A-chain mRNA on neointima formation in rat carotid artery after balloon injury and evaluated specificity of this ribozyme for PDGF A-chain mRNA by microarray analysis. The ultimate goal is to develop a gene therapy for treatment or prevention of coronary artery restenosis after PTCA.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
Our investigation confirmed to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.1 Synthesis of chimeric DNA–RNA hammerhead ribozyme
We used a 38-base chimeric DNA–RNA hammerhead ribozyme in which ribonucleotides at non-catalytic residues were replaced with deoxyribonucleotides and with phosphorothioate linkages at the 3' terminus for cleavage at the GUC sequence at nucleotide 921 in the loop structure of the rat PDGF A-chain mRNA [11]. A mismatch ribozyme with a single base change in the catalytic loop region was designed for use as a control (Fig. 1). The chimeric DNA–RNA hammerhead ribozyme and mismatch ribozyme were synthesized with a DNA–RNA synthesizer and purified by high performance liquid chromatography.


Figure 1
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  		Fig. 1 Structures of (A) chimeric DNA–RNA hammerhead ribozyme specific rat platelet-derived growth factor (PDGF) A-chain and (B) mismatch ribozyme. The chimeric DNA–RNA hammerhead ribozyme is 38-base nucleic acid. The catalytic part is composed only of ribonucleotides, whereas the other portion contains deoxyribonucleotides and two phosphorothioate linkages at the 3' terminus to improve stability. The mismatch ribozyme contains a single altered base (A to C) in the catalytic loop region.

 
2.2 Vascular injury and treatment with ribozyme
Male Wistar rats (Charles River Breeding Laboratories, Shizuoka, Japan) weighing 300 to 350 g were used in all experiments. Rats were anesthetized by intraperitoneal injection of pentobarbital. A midline incision was made in the neck to expose both carotid arteries. A 2F embolectomy catheter (Baxter Healthcare Corp., Irvine, CA, USA) was introduced into the left common carotid artery, and the balloon was inflated with saline and drawn toward the arteriotomy site five times to produce a distending and de-endothelializing injury [15].

For each in vivo transfer, ribozyme or mismatch ribozyme was diluted to 2 or 5 µg in 50 µl of saline, and 4 or 10 µl of 20 kDa polyethylenimine reagent [16] was diluted in 50 µl of saline. After standing at room temperature for 45 min, the two reagents were combined and incubated at room temperature for 15 min. The combined reagents were administered and incubated in the lumen of the injured carotid artery for 10 min.

To assess the distribution of ribozyme in the carotid artery after balloon injury, 5 µg of FITC-labeled ribozyme was incubated within the lumen for 10 min before the balloon injury. Vessels were then harvested at 30 min, 2 h, and 24 h after transfection and fixed in 4% paraformaldehyde. Sections were examined by fluorescence microscopy.

2.3 Morphometric analysis of intimal thickening
To assess the effects of ribozyme on neointima formation, rats were killed at 14 days after balloon injury, with sodium pentobarbital (intraperitoneal injection, 100 mg/kg body weight) perfused with saline, after then perfused with 10% formalin at physiological pressure. For immunohistochemistry and morphometric analysis, carotid arteries were fixed in 100% methanol overnight, and then the middle third of the common carotid artery was cut into four segments and embedded in paraffin. Specimens were cross-sectioned at a thickness of 3 µm and stained with hematoxylin and eosin. The intimal and medial cross-sectional areas of four cross sections of the artery from each rat were measured. The intima/media cross-sectional area ratios were determined with a computerized apparatus and NIH Image software (version 1.57).

2.4 Extraction of mRNA from injured vessel
Untreated injured vessel or injured vessel treated with the ribozyme was removed and washed with ice-cold-RNase free phosphate-buffered saline (PBS). mRNA was extracted directly with oligo-dT-cellulose using the Quick Prep Micro mRNA Purification Kit (Amersham Pharmacia Biotech, Buckinghamshire, UK) in accordance with the instruction by the manufacturer.

2.5 RT-PCR analysis for PDGF A-Chain mRNA
RT-PCR was performed as described previously [17]. Briefly, aliquots of mRNA were reverse-transcribed into single-stranded cDNA. Sense primer (5'-AAGCATGTGCCGGAGAAGCG-3') and antisense primer (5'-TCCTCTAACCTCACCTGGAC-3') flanking the GUC cleavage site were used for PCR amplification of rat PDGF-A chain mRNA to yield a 305-base pair (bp) product. Sense primer (5'-TCAAGAACGAAAGTCGGAGG-3') and antisense primer (5'-GGACATCTAAGGGCATCACA-3') for rat 18S ribosomal RNA was used as an internal control to yield a 488-bp product. PCR was performed in an automatic thermocycler (Perkin–Elmer, Foster, CA, USA). After initial denaturation at 96 °C for 5 min, PCR amplification consisted of 30 cycles of denaturation at 94 °C for 1 min, annealing at 58 °C for 2 min, and primer extension at 72 °C for 1 min, followed by a final extension at 72 °C for 10 min. PCR products were separated by electrophoresis on 1.5% agarose gels.

2.6 Western blot analysis of PDGF-AA protein
Injured vessel treated with or without ribozyme was removed, washed with ice-cold PBS, and homogenized to a uniform suspension in 500 µl of lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.02% sodium azide, 100 µg/ml phenylmethylsulfonyl fluoride, 1 µg/ml aprotinin, 1% Triton-X). Samples were sonicated for 2 min, then centrifuged at 16 000 g at 4° for 10 min, mixed with 400 µl methanol and 100 µl chloroform, and sonicated again for 2 min. After centrifugation at 16 000 g at 4° for 10 min, the supernatants were evaporated. They were suspended in 100 µl of PBS and sonicated for 4 min. The concentration of protein was determined by the method of Lowry et al. [18].

For Western blotting 30 µl of each tissue extract was mixed with 30 µl of sample buffer [63 mM Tris–HCl (pH 6.8), 2% SDS, 10% glycerol, 5% 2-mercaptoethanol]. Samples were boiled for 90 s and subjected to 10% polyacrylamide gel electrophoresis. The proteins were transblotted on to nitrocellulose, and the membranes were incubated with rabbit polyclonal antibody specific for PDGF-AA (Austral Biologicals, San Ramon, CA, USA) or the mouse monoclonal antibody specific for {alpha}-tubulin as a control (Sigma BioScience, St. Louis, MO, USA) diluted 1:500 in 5% non-fat milk in TBST solution (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, 0.05% Tween-20) for 3 h at room temperature. Membranes were then incubated with goat anti-mouse IgG for 1 h at room temperature, and then washed once with TBST for 15 min. and then four times for 5 min. Immune complexes on the membrane were detected with an ECL kit (Amersham Pharmacia Biotech).

2.7 Microarray analysis
Experiments with GeneChips (Affymetrix, Santa Clara, CA) were performed according to the manufacturer's instructions [19,20]. Double-stranded cDNA was synthesized by reverse transcription performed with the Superscript Choice System (Gibco Life Technologies, Gaitherburg, MD, USA) from mRNA extracted from the injured vessel. The resulting cDNA was purified with phenol/chloroform extraction with Phase Lock Gel (5 Prime to 3 Prime, Inc. Boulder, CO) and concentrated by ethanol precipitation. Synthetic double-stranded cDNA transcription into biotin-labeled cRNA was done in vitro with a MEGAscript T7 kit (Ambion, Austin, Texas). Biotin-labeled cRNA was then isolated with an RNeasy Mini Kit (Qiagen, Tokyo, Japan) and precipitated with ethanol. The cRNA was fragmented to 50 to 200-nucleotide pieces as described by Wodicka et al. [21]. After fragmenting, 5 µg of cRNA was injected into an R-U34 probe array cartridge (Affymetrix), which contains probe sets for 970 rat genes for hybridization. U34 array contains oligonucleotides representing transcripts of metabolic enzymes, growth factors and receptors, kinases and phosphatases, nuclear receptors, transcription factors, DNA damage repair genes, apoptosis genes, stress response genes, membrane proteins, and cell-cycle regulators. Probe arrays were treated with biotinylated anti-streptavidin goat antibody (Vector Laboratories, Burlingame, CA, USA) and stained with streptavidin phycoerythrin (Molecular Probes, Eugene, OR, USA). Probe arrays were scanned twice with a GeneChip scanner at a resolution of 3 µm. The intensity for each feature of the array was captured with Affymetrix GeneChip Expression Analysis Software according to standard Affymetrix procedures, and the gene expression data were analyzed with Microsoft Excel. Expression values of transcript in different groups were normalized by adjusting GAPDH expression as 5000 to the same value. The final values for each transcript as increments was the mean of duplicate values for injured vessel minus the average values for non-injured vessel.

2.8 Statistical analysis
Values are reported as mean±S.E.M. Statistical analysis was done with Student's t-test for unpaired data or with two-way analysis of variance (ANOVA) or Duncan's multiple range test.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Time course of PDGF A-chain mRNA expression in injured vessel
PDGF A-chain mRNA was not detected in rat carotid artery prior to balloon injury. Expression of PDGF A-chain mRNA increased from 2 to 72 h after injury (Fig. 2).


Figure 2
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  		Fig. 2 Time course of expression of platelet-derived growth factor (PDGF) A-chain mRNA in rat carotid artery after balloon injury. mRNA was extracted directly with oligo-dT-cellulose, and expression of PDGF A-chain mRNA was evaluated by reverse transcription and polymerase chain reaction of RNA from vessels before and 2, 6, 12, 24 h, and 7 days after balloon injury. 18S ribosomal RNA is included as an internal control. The ratio of the abundance of PDGF A-chain mRNA to that of 18S mRNA was evaluated by densitometric analysis. Data are the means of experiments carried out in duplicate.

 
3.2 Distribution of ribozyme in injured vessel
Distribution of FITC-labeled chimeric DNA–RNA ribozyme targeting PDGF A-chain mRNA in carotid artery after balloon injury is shown in Fig. 3. FITC-labeled ribozyme was localized in the midlayer of smooth muscle in the carotid artery by 30 min after injury, and levels increased until 24 h after balloon injury. No fluorescence was seen in the control arteries. FITC-labeled mismatch ribozyme was localized in the midlayer of smooth muscle in the carotid artery similar to FITC-labeled ribozyme (results not shown).


Figure 3
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  		Fig. 3 Uptake of fluorescein–isothiocyanate (FITC)-labeled chimeric DNA–RNA hammerhead ribozyme against platelet-derived growth factor (PDGF) A-chain into carotid artery after balloon injury. Five micrograms of FITC-labeled ribozyme was incubated within the artery lumen for 10 min. Vessels were harvested 30 min, 2 h, and 24 h after transfection and perfusion-fixed with 4% paraformaldehyde. Sections were examined by fluorescence microscopy.

 
3.3 Effects of ribozyme on neointima formation
Neointima formations in rat carotid artery treated with chimeric DNA–RNA ribozyme targeting PDGF A-chain mRNA are shown in Fig. 4. Two and 5 µg of ribozyme significantly (P<0.05) reduced neointima formation to 44 and 55%, respectively, compared to neointima formation in arteries not treated with ribozyme. Mismatch ribozyme did not affect neointima formation in rat carotid artery.


Figure 4
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  		Fig. 4 Effect of chimeric DNA–RNA ribozyme against platelet-derived growth factor (PDGF) A-chain on neointima formation in rat carotid artery 2 weeks after balloon injury. Two or 5 µg of ribozyme or mismatch ribozyme was incubated within the artery lumen for 10 min. (A) Specimens were cross-sectioned at 3 µm and stained with hematoxylin and eosin. (B) Intimal and medial cross-sectional areas of four cross sections of artery obtained from each rat were measured. The intima/media cross-sectional area ratios were determined. Data are the mean±S.E.M. (n=4). * P<0.05 vs. balloon injury without ribozyme:Ribozyme (–).

 
3.4 Effects of ribozyme on expression of PDGF A-chain in injured vessel
The effects of 5 µg of chimeric DNA–RNA ribozyme targeting PDGF A-chain mRNA on expression of PDGF A-chain mRNA and production of PDGF-AA protein in injured vessel at 6 h after balloon injury are shown in Fig. 5. Levels of PDGF A-chain mRNA and PDGF-AA protein were significantly higher in injured vessel in comparison with those in normal vessel. Ribozyme markedly reduced levels of PDGF A-chain mRNA and PDGF-AA protein in injured vessel. Mismatch ribozyme did not affect expression of PDGF A-chain mRNA or production of PDGF-AA protein in injured vessel.


Figure 5
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  		Fig. 5 Effect of chimeric DNA–RNA ribozyme specific for platelet-derived growth factor (PDGF) A-chain on expression of PDGF A-chain mRNA and PDGF-AA protein in carotid artery after balloon injury. Two or 5 µg of or mismatch ribozyme was incubated within the artery lumen for 10 min. (A) Expression of PDGF A-chain mRNA in the carotid artery 6 h after balloon injury. (B) Ratio of PDGF A-chain to 18S mRNA. Data are the mean±S.E.M. (n=4). * P<0.01 vs. balloon injury without ribozyme:Ribozyme (–). (C) Expression of PDGF-AA protein in carotid artery 24 h after balloon injury. {alpha}-Tubulin is included and used as an internal control.

 
3.5 Microarray analysis of injured vessel
To determine which transcripts increased in injured vessel and to assess the specificity of the chimeric DNA–RNA ribozyme targeting PDGF A-chain mRNA, we performed microarray analysis. The levels of the 60 most abundant transcripts in injured vessel are shown in Table 1. The transcript in injured vessel was FLK-1 followed by interleukin-1 receptor-related protein, metallothionein-2 and methallothionein-1, E-crystallin, DNA polymerase, retinoic acid receptor {alpha}2 isoform (RAR), heat shock protein, ubiquitin conjugating enzyme, clone par-4 induced by effectors of apoptosis, mitogen-activated protein (MAP) kinase, Fas antigen, G6Pase, PI-5-P-kinase, p38 MAP kinase, PCNA, TGF-{alpha}1, PDGF A-chain, ERK3, hydroxysteroid sulfotransferase subunit, ERK1, Arnt1, and interleukin converting enzyme-like cysteine protease, etc. Levels of 525 transcripts were increased in injured vessel. Of these, 170 were expressed sequence tags (EST). The injured vessels expressed genes of metabolic enzymes, growth factors, cytokines, cell cycle regulators and transcription factors abundantly.


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  		Table 1 Sixty most abundant transcripts in injured carotid

 
Increments in transcript of cytokines, growth factors, and transcription factors analyzed by microarray in injured vessel treated with or without 5 µg of ribozyme are shown in Fig. 6. Among these transcripts, only the increase in levels of PDGF A-chain transcript was inhibited completely by treatment with ribozyme. Increments in transcripts of intracellular signaling systems, kinases, and cell cycle-related peptides are shown in Fig. 7, and increments in transcripts of metabolic enzymes as analyzed by microarray in carotid artery 6 h after balloon injury and are shown in Fig. 8. Treatment with ribozyme completely inhibited increases in expression of PCNA/cyclin, cyclin B, calcium-dependent tyrosine kinase, cdc 42-binding kinase, serine/threonine kinase {gamma}-PAK transcripts and markedly inhibited increases expression of in MAP kinase kinase, ERK3, ERK1, and cdc2-related protein kinase. Among transcripts of metabolic enzymes, the increases in prostacyclin synthase, interleukin 1β converting enzyme, G6Pase were completely inhibited, and the increases in DNA topoisomerase II, DNA polymerase IIB, and interleukin 1-converting enzyme-related protease CP P32 transcripts were markedly inhibited by treatment with ribozyme.


Figure 6
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  		Fig. 6 Increments in transcript of cytokines, growth factors, and transcription factors analyzed by microarray (R-U34 GeneChip Array, Affymetrix) in carotid artery 6 h after balloon injury treated with (closed column) or without (open column) 5 µg of chimeric DNA–RNA ribozyme to platelet-derived growth factor (PDGF) A-chain. Final transcript values are means of duplicate values in injured vessel minus the average values of non-injured vessel.

 

Figure 7
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  		Fig. 7 Increments in transcript of intracellular signaling systems, kinases, and cell cycle-related peptides analyzed by microarray (R-U34 GeneChip Array, Affymetrix) in carotid artery 6 h after balloon injury treated with or without 5 µg of chimeric DNA–RNA ribozyme to platelet-derived growth factor (PDGF) A-chain. Final transcript values are means of duplicate values in injured vessel minus the average values for non-injured vessel.

 

Figure 8
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  		Fig. 8 Increments in transcript of metabolic enzymes analyzed by microarray (R-U34 GeneChip Array, Affymetrix) in carotid artery 6 h after balloon injury treated with or without 5 µg of chimeric DNA–RNA ribozyme to platelet-derived growth factor (PDGF) A-chain. Final transcript values are means of duplicate values for injured vessel minus the average values for non-injured vessel.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Successful applications of ribozymes for inhibition of expression for HIV1 and cancer-associated genes have been reported [22,23]. In the field of cardiovascular medicine, several lines of research have suggested that gene therapy may be applicable to most cardiovascular diseases, including arterial proliferative diseases such as atherosclerosis, arterial restenosis after angioplasty, and hypertension.

In the present study, the chimeric DNA–RNA hammerhead ribozyme targeting PDGF A-chain mRNA effectively reduced neointima formation in rat carotid artery after balloon injury. The ribozyme completely inhibited expression of PDGF A-chain mRNA and production of PDGF-AA protein in injured vessels, suggesting that the ribozyme inhibits neointima formation through degradation of PDGF A-chain mRNA. Recently, it was reported that VSMCs in the neointima are from circulating bone marrow cells not midlayer VSMCs [24]. Intimal VSMCs with the synthetic phenotype produce high levels of several cytokines and growth factors including PDGF, TGF-β, bFGF, endothelin, and angiotensin II, that are involved in neointima formation [25]. The neointima includes VSMC and extracellular matrix [26]; therefore, it is likely that the ribozyme to PDGF A-chain partially, but not entirely, inhibited neointima formation.

One significant problem in using ribozymes as gene therapy is degradation. RNA ribozymes are degradated in culture medium and in living cells, and diminishes availability of the ribozyme [27]. For application of ribozymes in tissues, high catalytic efficiency, stability, and adequate levels of ribozyme are necessary. A number of modifications can improve stability, specificity, and efficacy of ribozymes. In the present study, we used a chimeric DNA–RNA hammerhead ribozyme in which deoxyribonucleotides were substituted for ribonucleotides at non-catalytic residues to improve catalytic activity and stability [28]. In addition, two deoxyribonucleotides at the 3'-terminal of the ribozyme were modified with phosphorothioate linkages to improve resistance to nucleases [29]. Moreover, liposome-complexed molecules are transported preferentially to the cytoplasm [30], whereas others proposed nuclear localization of the delivered products [31]. In the present experiments, FITC-labeled chimeric DNA–RNA ribozyme introduced with polyethylenimine was taken up by the midlayer vascular smooth muscle of the carotid artery beginning at 30 min and continuing 24 h after balloon injury. The time course of the uptake of ribozyme corresponds to the increase in PDGF A-chain expression in carotid artery after balloon injury. Thus the ribozyme was relatively stable and was taken up by vascular smooth muscle and hybridized with the increased PDGF A-chain mRNA. It appears that polyethylenimine, in addition to assisting ribozyme delivery to cells, protects the ribozyme from degradation by nucleases.

Recently, ribozymes targeting c-myc [32], TGF-β1 [33], and leukocyte-type 12-lipoxygenase [34] were reported to inhibit efficiently neointima formation in vivo. These reports suggest that ribozymes may be effective gene therapies for restenosis after PTCA.

On the other hand, cytotoxic (thymidine kinase [35] and Fas ligand/p35 [36]), and cytostatic (retinoblastoma [37], p21 [38], and Gax [39]) gene therapies have been reported to inhibit the neointima formation effectively. However, these are therapy using genes, not the nucleic acid-based gene therapies. The nucleic acid composed with short nucleotides is easily designed to the target genes and may not have considerable side effects as the gene-induced secondary effects.

Another benefit of nucleic acid-based gene therapies, such as with antisense ODN and ribozyme, is the targeted inhibition of a specific target gene in a tissue. Antisense ODN inhibit target gene expression primarily by forming hybrids with the corresponding mRNA and causing depletion of the respective protein [40]. Antisense ODN also trigger RNase H-mediated degradation of the target mRNA [41] and interfere with processing of the pre-mRNA [42]. In contrast, ribozymes hybridize with and cleave the target RNA. Once the target is cleaved, the ribozyme can dissociate from the cleavage products and repeat the process with another RNA molecule [9]. Thus, ribozymes do not require cellular components and are highly specific in their inhibition of target gene expression. To evaluate the specificity of our engineered ribozyme targeting PDGF A-chain mRNA, we examined levels of specific transcripts in injured vessels with microarray technology.

PDGF A-chain mRNA levels increased considerably from 2 h after balloon injury in the present experiments. Possible compensatory changes in many transcripts could be also observed after balloon injury. Therefore the effects of the ribozyme on PDGF A-chain transcripts were evaluated by microarray with RNA isolated from vessel at 6 h after balloon injury. In the microarray analysis, levels of 525 transcripts were increased in injured vessel. Injured vessel expressed significant levels of transcripts for metabolic enzymes, growth factors, cytokines, cell cycle regulators, and transcription factors. The ribozyme targeting PDGF A-chain mRNA inhibited expression of only PDGF A-chain mRNA completely, suggesting a specific inhibitory effect of the ribozyme on PDGF A-chain mRNA.

Among the increases in transcripts of intracellular signaling systems, kinases, and cell cycle-related peptides, the increases in PCNA/cyclin, cyclin B, calcium-dependent tyrosine kinase, cdc42-binding kinase, serine/threonine kinase β-PAK in the injured vessel were completely inhibited, and the increases in MAP kinase kinase, extracellular signal-regulated kinase 3 (ERK3), ERK1, cdc2-related protein kinase transcripts were markedly inhibited by treatment of the chimeric DNA–RNA hammerhead ribozyme targeting PDGF A-chain mRNA. Among transcripts of metabolic enzymes, the increases in transcripts of prostacyclin synthase, interleukin-1β converting enzyme, G6Pase were completely inhibited, and the increases in DNA topoisomerase II, DNA polymerase IIB, and interleukin-1β converting enzyme-related protease CP P32 transcripts were considerably inhibited by the ribozyme. Since after the inhibition of the neointima formation by the ribozyme, cell cycles, metabolisms and phenotype of VSMC are also suppressed, the suppression of expression of transcripts for intracellular signaling systems, kinases, and cell cycle-related peptides may be secondary changes corresponding to the inhibition of neointima formation by the ribozyme.

In conclusion, our chimeric DNA–RNA hammerhead ribozyme targeting PDGF A-chain mRNA efficiently inhibited neointima formation in rat carotid artery after balloon injury with specific inhibition of PDGF A-chain mRNA expression. Our results suggest that this ribozyme may be useful for treatment of coronary artery restenosis after PTCA.

Time for primary review 25 days.


   Acknowledgements
 
This work was supported by a grant from the Ministry of Education, High-Tech Research Center Japan.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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