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Cardiovascular Research 2000 48(1):138-147; doi:10.1016/S0008-6363(00)00157-7
© 2000 by European Society of Cardiology
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Copyright © 2000, European Society of Cardiology

DNA–RNA chimeric hammerhead ribozyme to transforming growth factor-β1 mRNA inhibits the exaggerated growth of vascular smooth muscle cells from spontaneously hypertensive rats

Jian Teng, Noboru Fukuda*, Wen-Yang Hu, Mari Nakayama, Hirobumi Kishioka and Katsuo Kanmatsuse

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

* Corresponding author. Tel.: +81-3-3972-8111; fax: +81-3-3972-1098 nhukuda{at}med.nihon-u.ac.jp

Received 4 November 1999; accepted 6 June 2000


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objective: The purpose of this study was to develop DNA–RNA chimeric hammerhead ribozyme against transforming growth factor-β1 (TGF-β1) mRNA as a gene therapy agent for arterial proliferative diseases. Methods: A 38-base hammerhead ribozyme against rat TGF-β1 mRNA, to produce cleavage at the GUC sequence at nucleotide 825 according to the secondary structure of rat TGF-β1 mRNA was designed. To enhance its stability, we synthesized a DNA–RNA chimeric ribozyme with two phosphorothioate linkages at the 3'-terminal. We also synthesized a mismatch ribozyme with single base change in the catalytic loop region as a control. These ribozymes were delivered into rat vascular smooth muscle cells (VSMC) from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats by lipofectin-mediated transfection, and their biological effects were investigated. Results: According to in vitro cleavage studies, the synthetic ribozyme can cleave the synthetic substrate RNA into two RNA fragments. Chimeric ribozyme significantly inhibited DNA synthesis in VSMC from SHR but not in cells from WKY rats. Mismatch ribozyme showed only a little effect on growth of VSMC from SHR. Chimeric ribozyme significantly inhibited proliferation of VSMC from SHR; in contrast, the proliferation of VSMC from WKY rats was significantly increased by this chimeric ribozyme. Mismatch ribozyme did not affect proliferation of VSMC from either rat strain. Chimeric hammerhead ribozyme to rat TGF-β1 dose-dependently inhibited TGF-β1 mRNA expression detected by reverse transcription and polymerase chain reaction analysis in VSMC from both rat strains. Chimeric hammerhead ribozyme to rat TGF-β1 also dose-dependently inhibited TGF-β1 protein production detected by Western blot analysis. Conclusions: The present results demonstrated that our designed DNA–RNA chimeric hammerhead ribozyme to TGF-β1 mRNA might be a useful gene therapy agent for hypertensive vascular diseases.

KEYWORDS Gene therapy; Growth factors; Hypertension; Cell culture/isolation


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
It has been reported that the exaggerated growth of cardiovascular organs observed in patients with essential hypertension as well as in spontaneously hypertensive rats (SHR) is not the result of high blood pressure, but a primary event, and that treatment for hypertension should focus on achieving regression of excessive vascular growth [1]. Cultured vascular smooth muscle cells (VSMC) from SHR and normotensive Wistar-Kyoto (WKY) rats have distinct growth phenotypes; VSMC from SHR show a higher specific growth rate, abnormal contact inhibition, and accelerated entry into S phase of the cell cycle [2]. SHR-derived VSMC also exhibit increased accumulation of TGF-β mRNA as well as increased activation of this growth factor [3]. TGF-β represents a family of proteins that regulate growth and development of cells. TGF-β acts to inhibit growth of most cell types, but it has a dual effect on VSMC growth, stimulating growth at high cell densities and suppressing it at low cell densities. This is reported to result from an interaction of TGF-β with distinct TGF-β receptor subtypes at different cell densities [4]. Our previous study demonstrated the role of TGF-β in the exaggerated growth of SHR-derived VSMC by showing that an antisense oligodeoxynucleotide complementary to TGF-β1 mRNA inhibits the growth of these cells [3].

We demonstrated distinct expression of TGF-β receptor subtypes in VSMC from SHR and WKY rats [5]. VSMC from SHR show increased specific binding to TGF-β that is the result of higher affinity and more binding sites compared to VSMC from WKY rats. These data indicate that VSMC from SHR constitutively express high affinity TGF-β receptors, which are associated with the exaggerated growth of VSMC from SHR [5]. Recently, we observed abnormal regulation of TGF-β receptors on VSMC from SHR by angiotensin II (Ang II); an increase in the expression of the TGF-β receptors by Ang II facilitates the ability of endogenous TGF-β to counteract the stimulatory effect of Ang II on growth in VSMC from WKY rats, whereas endogenous TGF-β induced by Ang II cannot counteract the growth-promoting action of Ang II in VSMC from SHR [6]. This abnormality may facilitate vascular proliferation and underlie the development of hypertensive vascular disease.

Ribozymes are RNA molecules that catalytically cleave a phosphodiester bond of their respective target RNAs in a sequence-specific manner, thereby inhibiting the expression of specific gene products. The smallest hammerhead ribozyme has been used most widely to cleave mRNAs and inhibit gene expression [7,8]. In the last 15 years, ribozymes have progressed from subjects of scientific study to potential therapeutic agents for treatment of both acquired and inherited diseases [9]. Ribozymes have been used successfully to inhibit gene expression in cancer [10] and human immunodeficiency virus [11] targets, demonstrating the promise of this methodology. However, ribozyme therapy for hypertension has not yet been reported. The current study was undertaken to develop DNA–RNA chimeric ribozyme against TGF-β1 mRNA as a gene therapy agent for hypertensive vascular diseases.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
Our investigation conformed 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 Design of hammerhead ribozyme targeting rat TGF-β1 mRNA
We used ‘GENETYX-MAC: Second structure and Minimum Free Energy’ to choose a GUC cleavage site from rat TGF-β1 mRNA sequence and to prevent a double strand structure at the cleavage site [12], and we designed a 38-base hammerhead ribozyme (5'-GAAUGUCUCUGAUGAGUCCUUGCGGACGAAACGUAUUG-3') that cleaves rat TGF-β1 cDNA at nucleotide 825 (Fig. 1).


Figure 1
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  		Fig. 1 Secondary structure of rat transforming growth factor-β1 (TGF-β1) mRNA (from nucleotide 701 to 900) from GENETYX-MAC and the cleavage site of GUC sequence indicated by arrow at the nucleotide 825.

 
2.2 Synthesis of all-RNA ribozyme and target RNA using T7 RNA polymerase
All RNA ribozyme and target RNA were synthesized using T7 RNA polymerase and synthetic DNA template as described previously [13]. For a fully active template DNA strand, a fragment containing the region from –17 to –1 of the class III T7 RNA polymerase promoter followed by the complement of the desired RNA sequence was designed. To create a transcription template, a 17-nt complementary fragment corresponding to nt –17 to –1 of the promoter was annealed to the promoter portion of the template strand (Fig. 2). To prepare an annealed template, equal molar concentrations of the two strands were mixed, heated at 90°C for 3 min, and cooled quickly on ice. For synthesis of RNA, 3 mg of annealed template was mixed with 6 µl of T7 RNA polymerase (50 U/µl, Biochemicals, Osaka, Japan), 5 µl of {alpha}-32P-CTP (3000 Ci/mM, New England Nuclear, DE, USA), 50 U of RNase inhibitor (Takara Biochemicals, Osaka, Japan) and 50 µl of transcription reaction buffer [40 mM Tris-HCl (pH 8.0), 0.5 mM rNTP, 8 mM MgCl2, 5 mM DTT, 2 mM sperimidine] and incubated at 37°C for 4 h.


Figure 2
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  		Fig. 2 Synthetic DNA template, promoter region base paired from –17 to –1. Transcription starts at +1. N indicates one of the four nucleotides.

 
Phenol:chloroform (1:1) was added, vortexed and centrifuged at 16 000 rpm for 30 s. The supernatant was transferred to a new tube, mixed with equal amount of 25:1 chloroform:isoamyl alcohol, vortexed and centrifuged at 16 000 rpm for 30 s. The supernatant was mixed with 200 ml of 100% ethanol and centrifuged at 16 000 rpm for 15 min. The RNA pellet was washed with 75% ethanol twice, evaporated, and dissolved in 5 µl of diethyl pyrocarbonate-treated water.

2.3 In vitro cleavage reaction
In vitro cleavage reactions were performed as described previously [14]. Annealing of the ribozyme targeting target RNA was done by combining 100 nM ribozyme and 10 nM target RNA in 20 µl of 50 mM Tris-HCl (pH 8.0), heating at 90°C for 1 min, and then cooling over 30 min to 37°C. The cleavage reaction was initiated by adding 2 µl of 250 mM MgCl2 to the annealed ribozyme and target RNA in cleavage reaction buffer [50 mM Tris-HCL (pH 8.0) and 25 mM MgCl2] and then incubating at 37°C for 1, 5, or 15 h. The reaction was stopped by adding 10 µl of bromophenol blue solution. Samples were heated at 90°C for 2 min and cooled quickly on ice. Five microliters of each sample was loaded onto a sequencing gel for electrophoresis. The gel was dried and exposed to film.

2.4 Designing chimeric DNA–RNA hammerhead ribozyme
For VSMC transfection, we designed a 38-base chimeric DNA–RNA hammerhead ribozyme in which ribonucleotides at noncatalytic residues were replaced with deoxyribonucleotides and with two phosphorothioate linkages at the 3' terminus for cleavage at the GUC sequence at nucleotide 825 of the rat TGF-β1 mRNA (Fig. 3A). A mismatch ribozyme with a single base change in the catalytic loop region was designed for use as a control (Fig. 3B). Chimeric DNA–RNA hammerhead ribozyme and mismatch ribozyme were synthesized with a DNA–RNA synthesizer and purified by high performance liquid chromatography.


Figure 3
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  		Fig. 3 Sequences of the DNA–RNA chimeric hammerhead ribozyme to rat transforming growth factor-β1 (TGF-β1) mRNA and a mismatch ribozyme with only one base differing in the catalytic loop region. The ribozyme (lower) cleaves the GUC codon of TGF-β1 mRNA (upper) as indicated by the arrow. Ribonucleotides of ribozymes are underlined. Phosphorothiate linkages are marked with a star.

 
2.5 Cell culture and establishment of quiescence
VSMC were obtained by explant method [15] from aortas of 8-week-old male SHR and WKY rats (SHR Corporation, Funabashi, Chiba, Japan) as described previously [2]. They were seeded and grown in Dulbecco's modified Eagle medium (DMEM) with 10% calf serum (Gibco Life Technologies, Inc. Gaitherburg, MD, USA), 100 U/ml penicillin and 100 mg/ml streptomycin. When the cells reached confluency in 7 to 10 days, they showed the hill-and-valley pattern that is typical of smooth muscle cells in culture. They were passaged by trypsinization with 0.1% trypsin (Gibco) in Ca2+- and Mg2+-free Dulbecco's phosphate-buffered saline (PBS) and seeded in 80-cm2 tissue culture flasks at a density of 105 cells/ml. Experiments were performed on VSMC after 3–5 passages. For experiments trypsinized cells were plated in 24-well culture dishes at a density of 5x104 cells/cm2 They were incubated in DMEM containing 10% calf serum for 24 h, and the culture medium was then changed to DMEM without calf serum. The cells were incubated in this medium for 24 h to establish quiescence.

2.6 Delivery of ribozyme into VSMC
For each transfection, chimeric DNA–RNA ribozyme was diluted to 0.01–1.0 µM in 100 µl of serum-free DMEM, and 5 µl of lipofectin reagent (Gibco) was diluted in 100 µl of serum-free DMEM. They stood at room temperature for 45 min and then combined and incubated at room temperature for 15 min. Quiescent VSMC were washed twice with serum-free DMEM, then 200 µl of lipofectin-ribozyme complexes was added to the cells and incubated at 37°C in a CO2 incubator [16].

2.7 Determination of DNA synthesis
Incorporation of [3H]thymidine into newly-synthesized DNA was performed as described previously [17]. Fresh DMEM containing 100 U/ml penicillin and 100 mg/ml streptomycin was added to 24-well cluster dishes containing quiescent VSMC. The agents to be tested were added for 20 h, and the medium was then changed to DMEM containing 0.5 µCi/ml of [3H]thymidine (NEN, Wilmington, DE, USA). [3H]Thymidine incorporation was terminated after 2 h by removal of the labeled medium. Each well was washed with 1 ml of iso-osmotic solution (150 mM NaCl) to remove excess [3H]thymidine, and the cells were fixed with 1 ml of ethanol:acetic acid (3:1) for 10 min. The cells were washed with 1 ml of H2O, acid-insoluble material was precipitated with 1 ml of 0.5 M ice-cold perchloric acid, and DNA was extracted into 1.5 ml of perchloric acid by heating at 90°C for 20 min. The perchloric acid containing solubilized DNA was transferred to scintillation vials, and the radioactivity was measured in a liquid scintillation counter.

2.8 Determination of cell numbers
VSMC were seeded in DMEM containing 5% calf serum in 24-well culture dishes at 5x104 cells/cm2 (105 cells/well) without or with test agents. The culture media was replaced every 24 h. Cells were harvested with 0.05% trypsin 24, 48 and 72 h after inoculation, and cell numbers were counted in a Coulter Counter (Coulter Electronics Ltd., Luton, Beds, UK).

2.9 Reverse transcription and polymerase chain reaction (RT-PCR) analysis of mRNAs encoding TGF-β1
Quiescent VSMC at a density of 105 cells/cm2 in 2-cm2 wells were washed with PBS and lysed in 800 µl of RNAzol B (Biotecx, Houston, TX, USA). Cell lysates were mixed with 80 µl of chloroform, incubated at 4°C for 15 min, and centrifuged at 12 000xg for 15 min to extract total RNA. A portion (300 µl) of each aqueous phase was mixed with an equal volume of isopropanol, incubated at –20°C for 45 min, and centrifuged at 12 000xg for 15 min at 4°C to precipitate the RNA. The RNA pellet was washed twice with 500 µl of 75% (v/v) ethanol by vortex mixing and centrifugation at 7500xg for 8 min at 4°C, dried, and dissolved in 10 µl of a solution containing 10 mM Tris-HCl (pH 8.0) and 1 mM EDTA by incubation for 15 min at 65°C. Each sample was treated with 0.5 u of DNase (Gibco) in 0.5 µl of DNase buffer [20 mM Tris-HCl (pH 8.3), 50 µM KCl, 2.5 mM MgCl2] at room temperature for 45 min, after which the DNase was inactivated by adding 0.5 µl of 20 mM EDTA and heating at 98°C for 10 min.

RT-PCR was performed as described previously [18]. Briefly, aliquots of RNA (1 µg/20 µl) were reverse-transcribed into single-stranded cDNA by incubation for 10 min at 30°C, 30 min at 42°C, and 5 min at 99°C in 20 µl containing 5 U of avian myeloblastoma virus reverse transcriptase (Life Sciences, St. Petersburg, FL, USA), 10 mM Tris-HCl (pH 8.3), 5 mM MgCl2, 50 mM KCl, 1 mM of each deoxynucleotide triphosphate, and 2.5 µM random hexamers. The diluted cDNAs (5 µl) were then subjected to PCR in a final volume of 25 µl containing 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 4 mM MgCl2, 0.625 U of Taq DNA polymerase (Takara) and 0.2 µM each of sense and antisense primers. Sense primer (5'-GCCCTGGATACCAACTACTGCT-3') and antisense primer (5'-AGGCTCCAAATGTAGGGGCAGG-3') flanking the GUC cleavage site were used for PCR amplification of TGF-β1 mRNA to generate a 161-base pair (bp) product as described previously [19]. Sense primer (5'-TCAAGAACGAAAGTCGGAGG-3') and antisense primer (5'-GGACATCTAAGGGCATCACA-3') for rat 18S ribosomal RNA were used as an internal control. PCR was performed in an automatic thermocycler (Perkin–Elmer Cetus, Norwalk, CT, USA). Because the amount of PCR product corresponding to each of the target mRNAs increased in a linear manner from 20 to 35 cycles, following initial denaturation at 96°C for 5 min, PCR amplification was performed using 30 cycles of denaturation for 60 s at 94°C, annealing for 60 s at 60°C, extension for 60 s at 72°C, and final single cycle of extension for 10 min at 72°C. The PCR products were separated by electrophoresis on 1.5% agarose gel and stained with ethidium bromide.

2.10 Western blot analysis of TGF-β1 protein in VSMC
Ribozyme-treated and untreated VSMC at a density of 105 cells/cm2 in 10 cm2 wells were washed with PBS and lysed in lysis buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 0.02% sodium azide, 100 mg/ml phenylmethylsulfonyl fluoride, 1 mg/ml aprotinin, 1% Triton X-100]. After centrifugation, protein in the supernatant was purified by precipitation with methanol and chloroform. The protein concentration was determined by the method of Lowry et al. [20]. Western blot analysis was performed on 5 µg of protein with 20 µl of sample buffer as described previously [21]. The samples were boiled for 3 min and subjected to 10% polyacrylamide gel electrophoresis by the method of Laemmli [22]. The proteins were then blotted onto nitrocellulose membrane (Bio-Rad Laboratories, Hercules, CA, USA) according to the method of Towbin et al. [23]. After washing in 10 mM Tris-HCl (pH 8.0) with 150 mM NaCl and 0.05% Tween-20 (TBST) briefly, the membrane was incubated with rabbit pan specific polyclonal antibody for TGF-β (R and D Systems Inc., Minneapolis, MN, USA) or monoclonal anti-{alpha}-tubulin (Sigma Biosciences, St. Louis, MO, USA) as a control diluted 500 fold (v/v) in 5% nonfat milk in TBST overnight at 4°C. The membranes were then incubated with goat anti-rabbit IgG horseradish peroxidase conjugate (1:300, Bio-Rad) or goat anti-mouse IgG horseradish peroxidase conjugate (1:300, Bio-Rad) in 10 mM NaH2PO4 in saline for 1 h at room temperature. After washing with TBST for 5 min three times, immune complexes on the membranes were visualized calorimetrically by incubation with 10 ml of 0.1 M Tris-HCl (pH 7.6) with 3 mg of diaminobenzidine-HCl and 2 µl of 30% H2O2.

2.11 Statistics
Values are given as mean SEM. Significance between the mean values was evaluated by Student's t-test for unpaired data and by two-way analysis of variance (ANOVA) followed by Duncan's multiple range test.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 In vitro cleavage reaction
In vitro cleavage reaction with a synthetic ribozyme targeting rat TGF-β1 mRNA and a synthetic target mRNA was performed as shown in Fig. 4. In the presence of MgCl2, synthetic ribozyme cleaved the synthetic target RNA into two fragments that were consistent with the predicted sizes (60 and 27 bases). In the absence of MgCl2, ribozyme did not cleave the target RNA.


Figure 4
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  		Fig. 4 In vitro cleavage reaction with synthetic ribozyme to rat transforming growth factor-β1 (TGF-β1) mRNA (R) and target RNA (T). Annealed ribozyme (100 nM) and target RNA (10 nM) were incubated for 1, 5 and 15 h in the presence (+) of 25 mM MgCl2 or for 15 h in the absence (–) of MgCl2 respectively. In an another experiment, annealed mismatch ribozyme (MmR, 100 nM) and target RNA (10 nM) were incubated for 15 h in the presence of 25 mM MgCl2.

 
3.2 Effects of ribozyme targeting TGF-β1 mRNA on growth of VSMC from WKY rats and SHR
The effects of chimeric DNA–RNA hammerhead ribozyme targeting rat TGF-β1 mRNA and mismatch ribozyme on basal DNA synthesis in quiescent VSMC from WKY rats and SHR is shown in Fig. 5A. Basal DNA synthesis in VSMC from SHR was higher than that in cells from WKY rats (P<0.05). Concentrations of 0.01 to 1.0 µM ribozyme significantly (P<0.01) inhibited basal DNA synthesis in VSMC from SHR. Concentrations of 0.1 and 1.0 µM mismatch ribozyme also significantly (P<0.05) inhibited DNA synthesis in VSMC from SHR. The inhibitions of basal DNA synthesis in VSMC from SHR with 0.1 and 1.0 µM ribozyme were significantly (P<0.01) larger than those with the same concentrations of mismatch ribozyme. Ribozyme did not affect DNA synthesis in VSMC from WKY rats.


Figure 5
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  		Fig. 5 Effects of DNA–RNA chimeric hammerhead ribozyme to transforming growth factor-β1 (TGF-β1) mRNA on basal DNA synthesis in vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats (open column) and spontaneously hypertensive rats (SHR) (closed column). (A) Quiescent VSMC were incubated without (control) or with 0.1 and 1.0 µM mismatch ribozyme or 0.01–1.0 µM ribozyme in the presence of lipofectin for 20 h. (B) Time course of ribozyme to TGF-β1 treatment on DNA synthesis in VSMC from WKY rats and SHR. Quiescent VSMC were incubated with 0.1 µM ribozyme in the presence of lipofectin for 1, 4, 12 and 24 h and compared to untreated (0 h) control cells. Then [3H]thymidine incorporation into DNA was determined. Data are presented as mean±SEM (n = 4). *P<0.05, **P<0.01 compared with control (in the absence of ribozyme). #P<0.01 compared with mismatch at same concentrations.

 
Time course of 0.1 µM ribozyme treatment on basal DNA synthesis in VSMC from WKY rats and SHR is shown in Fig. 5B. From 1 to 24 h, ribozyme significantly (P<0.05) inhibited basal DNA synthesis in VSMC from SHR in a time-dependent manner, whereas ribozyme only inhibited basal DNA synthesis in VSMC from WKY rats at 24 h (P<0.05).

Effects of chimeric DNA–RNA hammerhead ribozyme targeting TGF-β1 mRNA on proliferation of VSMC from WKY rats and SHR in the presence of 5% calf serum are shown in Fig. 6. Ribozyme targeting TGF-β1 significantly (P<0.05) increased the numbers of VSMC from WKY rats at 24, 48, and 72 h after inoculation. Ribozyme targeting TGF-β1 significantly (P<0.05) decreased the numbers of VSMC from SHR at 24, 48, and 72 h after inoculation. Mismatch ribozyme did not affect the numbers of VSMC from either rat strains.


Figure 6
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  		Fig. 6 Effects of DNA–RNA chimeric hammerhead ribozyme to transforming growth factor-β1 (TGF-β1) mRNA on proliferation of vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). VSMC were inoculated at 5x104 cells/cm2 in DMEM containing 5% calf serum and 0.1 µM ribozyme or mismatch ribozyme. Cells were replenished with DMEM containing 5% calf serum and 0.1 µM ribozyme or mismatch ribozyme every 24 h. They were trypsinized at 24, 48 and 72 h after inoculation, and cell number was determined in a Coulter Counter. Vertical bars represent mean±SEM (n = 4). *P<0.05, **P<0.01 compared with control (without ribozyme) #P<0.05 compared with mismatch ribozyme.

 
3.3 Effects of chimeric DNA–RNA hammerhead ribozyme targeting TGF-β1 mRNA and mismatch ribozyme on expression of TGF-β1 mRNA in VSMC from WKY rats and SHR
Expression of TGF-β1 mRNA in VSMC from WKY rats and SHR treated with 0.01–1.0 µM ribozyme targeting TGF-β1 or 0.1 and 1.0 µM mismatch ribozyme for 4 h is shown in Fig. 7. Levels of TGF-β1 mRNA were greater in VSMC from SHR than levels in cells from WKY rats. Concentrations of 0.01 to 1.0 µM ribozyme dose-dependently reduced levels of TGF-β1 mRNA in VSMC from both rat strains compared to the effects of mismatch ribozyme.


Figure 7
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  		Fig. 7 Effects of DNA–RNA chimeric hammerhead ribozyme to transforming growth factor-β1 (TGF-β1) on TGF-β1 mRNA expression in vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Quiescent VSMC were incubated for 4 h with 0.1 and 1.0 µM mismatch ribozyme or 0.01–1.0 µM ribozyme with lipofectin. The amounts of TGF-β1 mRNA and rat 18S ribosomal RNA were determined by reverse transcription-polymerase chain reaction. The positions of molecular size standards (in base pairs) are indicated in lane M.

 
3.4 Effects of chimeric DNA–RNA hammerhead ribozyme targeting TGF-β1 mRNA on expression of TGF-β1 protein in VSMC from WKY rats and SHR
Fig. 8 shows productions of TGF-β1 protein in VSMC from WKY rats and SHR treated with ribozyme targeting TGF-β1 or mismatch ribozyme for 20 h. Concentrations of 0.01 to 1.0 mM ribozyme significantly (P<0.05) reduced production of TGF-β1 protein in VSMC from SHR, and 0.1 and 1.0 mM ribozyme significantly (P<0.05) reduced production of TGF-β1 protein in VSMC from WKY rats. Mismatch ribozyme did not affect production of TGF-β1 protein in VSMC from either rat strains.


Figure 8
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  		Fig. 8 Effects of DNA–RNA chimeric hammerhead ribozyme to transforming growth factor-β1 (TGF-β1) mRNA on expression of TGF-β1 protein in vascular smooth muscle cells (VSMC) from Wistar-Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Quiescent VSMC were incubated for 20 h with 0.1 and 1.0 µM mismatch ribozyme or 0.01–1.0 µM ribozyme with lipofectin. (A) The amounts of TGF-β1 and {alpha}-tubulin proteins were determined by Western blot analysis. (B) The ratio of TGF-β1 and {alpha}-tubulin proteins were evaluated by densitometric analysis. Data are presented as mean±SEM (n = 4). #P<0.05 compared with WKY rats, *P<0.05, **P<0.01 compared with control (without ribozyme).

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
TGF-β, a multifunctional modulator of cell metabolism and growth in vitro, acts as a growth inhibitor on most cell types [24] but has a dual effect on growth of VSMC, suppressing it at low cell densities and stimulating it at high cell densities [25]. This dual effect on growth of VSMC results from the differential interactions of TGF-β with different receptors at different cell densities [4]. TGF-β1 mRNAs accumulate to a greater extent in VSMC from SHR than in cells from WKY rats, and the role of TGF-β in the exaggerated growth of VSMC from SHR was confirmed by the inhibition of TGF-β by antisense DNA complementary to the corresponding mRNA [3]. Previously we demonstrated that VSMC from WKY rats and SHR differ in their expression of TGF-β receptor subtypes [5]. Moreover, we recently observed abnormal regulation of TGF-β receptors on VSMC from SHR by Ang II, by which endogenous TGF-β induced by Ang II can not counteract the growth-promoting action of Ang II in VSMC from SHR [6]. We observed that an Ang II type 1 receptor antagonist inhibits exaggerated growth of VSMC from SHR [26] and recently confirmed that endogenous Ang II is produced in a homogeneous culture of VSMC from SHR but not in cells from WKY rats. Expression of cathepsin D and ACE also increased [27]. In addition, we demonstrated that enhanced generation of Ang II is involved in the increased expression of TGF-β1 mRNA [28]. Our previous observations indicate that the increases in TGF-β and the abnormal regulation of TGF-β receptors by endogenous Ang II contribute to the exaggerated growth of VSMC from SHR. Therefore, TGF-β is the growth factor responsible for the exaggerated growth of VSMC in hypertension and is a potential target for gene therapy for hypertension. It seems to be more feasible that a therapy could be designed to prevent the organ damage due to hypertension and not so much the hypertension itself.

Ribozymes hybridize to a complementary RNA sequences and site-specifically cleave phosphodiester bonds in those sequences [29]. Whereas antisense DNA inhibits expression of the target gene primarily by formation of hybrids with pre-mRNA or mRNA to inhibit its processing or translation specificically [30]. However, antisense DNA also triggers RNase H-mediated degradation of the target RNA and interferes with the processing of pre-mRNA non-specificically [31]. Thus, ribozyme, in contrast to antisense DNA, can suppress gene expressions specifically.

In the present study, the synthetic hammerhead ribozyme had potent RNA catalytic activity in vitro. Under in vitro experimental conditions, the secondary structure of the target RNA or ribozyme is important. Since ribozyme cleaves the target RNA first by binding the complementary sequence in the target RNA, if the secondary structure of the target RNA or ribozymes affects binding of ribozyme and target, ribozyme activity is reduced [32]. Although in vitro studies of ribozyme targeting TGF-β1 are essential for quantitating its catalytic efficiencies, it can not be used to predict the efficiency of ribozymes when expressed in cells, because of the influence of the different expressed genes and multiple factors. Factors that may affect ribozyme activity in the cell include expression level, colocalization of ribozyme with the target RNA, genomic location of ribozyme gene relative to target gene, target site characteristics, secondary structure of the target RNA or ribozyme, cofactors or inhibitors such as proteins [33], Mg2+ concentration [34], temperature and pH [35]. Also, if ribozyme expression is not efficient, the level of ribozyme may be too low to cleave sufficient target RNA to produce a biological effect.

Cellular delivery of ribozyme is of crucial importance to inhibit gene expression using this method. The lipofectin agent we used here can interact spontaneously with DNA, fuse with cultured cells, and facilitate delivery of functional DNA to the cell. The technique is simple, reproducible, and more efficient than other commonly-used procedures [16]. Reports have already suggested that lipofectin-complexed molecules are transported preferentially to the cytoplasm. Previously we focused on chimeric DNA–RNA hammerhead ribozyme targeting PDGF A-chain mRNA. Fluorescein isothiocyanate-labeled ribozyme was delivered efficiently into cytosol and nuclei in VSMC after transfection with lipofectin-ribozyme complexes, indicating that chimeric ribozyme is relatively stable in culture medium and is taken up very quickly by VSMC. Another problem that arises from exogenous delivery is low stability due to rapid degradation of oligoribonucleotides in cells [36]. To enhance catalytic turnover and stability we synthesized chimeric DNA–RNA ribozyme with deoxyribonucleotides substituted for ribonucleotides at noncatalytic residues [35,37]. In addition, two deoxyribonucleotides at the 3' terminus of chimeric ribozyme were modified with phosphorothioate linkages to improve resistance to nucleases [38]. In the present experiments, both chimeric DNA–RNA ribozyme and mismatch ribozyme inhibit growth of VSMC especially those from SHR; however, the extent of inhibition by chimeric DNA–RNA ribozyme of DNA synthesis in VSMC from SHR was significantly greater than that by mismatch ribozyme, suggesting that a part of the effect of the ribozyme is mediated by a non-specific effect of DNA, because the chimeric DNA–RNA ribozyme contains deoxyribonucleotides. Concerning the mechanisms underlying the non-specific effects of DNA on cell growth, several studies have reported that charged DNA behaves like polyanions such as heparin and heparan, which bind and sequester heparin-binding growth factors, such as basic fibroblast growth factor or PDGF, at the basement membrane [39]. Another report described the nonspecific cellular activation of the transcription factor Sp1 by phosphorothioate-linkaged DNA [40].

In the present experiments, chimeric DNA–RNA hammerhead ribozyme targeting TGF-β1 mRNA efficiently inhibited exaggerated growth of VSMC from SHR but did not inhibit growth of cells from WKY rats. These results indicate that TGF-β is involved in an autocrine/paracrine manner in the exaggerated growth of VSMC from SHR. The ribozyme shows distinct effects on proliferation of VSMC from WKY rats and SHR; ribozyme increases proliferation of VSMC from WKY rats, whereas it reduces proliferation of VSMC from SHR. This result can be explained by the distinct expression of TGF-β receptor subtypes on VSMC from WKY rats and SHR.

TGF-β is involved in the pathogenesis of hypertensive vascular diseases, restenosis of the coronary artery after angioplasty [41] and glomerulonephritis [42]. The ribozyme targeting TGF-β1 mRNA that we describe here could be used for efficient gene therapy for such cardiovascular diseases.

In conclusion, the present study demonstrates that our chimeric DNA–RNA hammerhead ribozyme targeting TGF-β1 mRNA is catalytically active in VSMC and efficiently inhibit the exaggerated growth of VSMC from SHR with suppression of TGF-β1 mRNA. This chimeric DNA–RNA hammerhead ribozyme can be used not only as a tool to investigate the role of TGF-β1 in other physiological systems, but it may also provide the basis for the development of a ribozyme-gene therapy for hypertensive vascular diseases.

Time for primary review 22 days.


   Acknowledgements
 
This work was financially supported in part by Grant-in Aid for the High-Tech Research Center and by Special Research Grants for the Development of Characteristic Education from the Japanese Ministry of Education, Science, Sports and Culture to Nihon University.


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

  1. Korner P., Bobik A., Angus J.A., Adams M.A., Friberg P. Resistance control in hypertension. J Hypertens (1989) 7(suppl_4):S125–134.[Web of Science]
  2. Hadrava A., Tremblay J., Hamet P. Abnormalities in growth characteristics of aortic smooth muscle cells in spontaneously hypertensive rats. Hypertension (1989) 13:589–597.[Abstract/Free Full Text]
  3. Fukuda N., Tremblay J., Hamet P. Participation of proteolysis and transforming growth factor β1 activation in growth of cultured smooth muscle cells from spontaneously hypertensive rats. Genet Hypertens (1992) 218:227–229.
  4. Goodman L.V., Majack R.A. Vascular smooth muscle cells express distinct transforming growth factor b receptor phenotypes as a function of cell density in culture. J Biol Chem (1989) 264:5241–5244.[Abstract/Free Full Text]
  5. Fukuda N., Kubo A., Izumi Y., Soma M., Kanmatsuse K. Characteristics and expression of transforming growth factor-β receptor subtypes on vascular smooth muscle cells from spontaneously hypertensive rats. J Hypertens (1995) 13:831–837.[CrossRef][Web of Science][Medline]
  6. Fukuda N., Hu W.Y., Kubo A., et al. Abnormal regulation of transforming growth factor-β receptors on vascular smooth muscle cells from spontaneously hypertensive rats by angiotensin II. Hypertension (1998) 31:672–677.[Abstract/Free Full Text]
  7. Haseloff J., Gerlach W.L. Simple RNA enzymes with new and highly specific endoribonuclease activities. Nature (1988) 334:585–591.[CrossRef][Medline]
  8. Marschall P., Thomson J.B., Eckstein F. Inhibition of gene expression with ribozymes. Cell Mol Neurobiol (1994) 14:523–538.[CrossRef][Web of Science][Medline]
  9. Rossi J.J. Therapeutic ribozymes, principles and applications. Bio Drugs (1998) 9:1–10.
  10. Sioud M., Natvig J.B., Forre O. Preformed ribozyme destroyed tumour necrosis factor mRNA in human cells. J Mol Biol (1992) 223:831–835.[CrossRef][Web of Science][Medline]
  11. Sarver M., Cantin E., Chang P., et al. Ribozymes as potential anti-HIV-1 therapeutic agents. Science (1990) 247:1222–1224.[Abstract/Free Full Text]
  12. Qian S.W., Kondaiah P., Roberts A.B., Sporm M.B. cDNA cloning by PCR of rat transforming growth factor-β1. Nucleic Acid Res (1990) 18:3059.[Free Full Text]
  13. Milligan J.F., Groebe D.R., Witherell G.W., Uhlenbeck O.C. Oligoribonucleotide synthesis using T7 RNA polymerase and synthetic DNA templates. Nucleic Acid Res (1987) 15:8783–8798.[Abstract/Free Full Text]
  14. Saxena S.K., Ackerman E.J. Ribozymes correctly cleave a model substrate and endogenous RNA in vivo. J Biol Chem (1990) 265:17106–17109.[Abstract/Free Full Text]
  15. Ross R. The smooth muscle cell-II. Growth of smooth muscle in culture and formation of elastic fibers. J Cell Biol (1971) 50:172–186.[Abstract/Free Full Text]
  16. Felgner P.L., Gadek T.R., Holm M., et al. Lipofection: A highly efficient, lipid-mediated DNA-transfection procedure. Proc Natl Acad Sci USA (1987) 84:7413–7417.[Abstract/Free Full Text]
  17. Franks D.J., Plamondon J., Hamet P. An increase in adenyl cyclase activity precedes DNA synthesis in cultured vascular smooth muscle cells. J Cell Physiol (1984) 119:41–45.[CrossRef][Web of Science][Medline]
  18. Mocharla H., Mocharla R., Hocks M.E. Coupled reverse transcription-polymerase chain reaction (RT-PCR) as a sensitive and rapid method for isozyme genotyping. Gene (1990) 93:271–275.[CrossRef][Web of Science][Medline]
  19. Qian X., Jin L., Grande J.P., Lloyd R.V. Transforming growth factor-β and p27 expression in pituitary cells. Endocrinology (1996) 137:3051–3060.[Abstract]
  20. Lowry O.H., Rosebrough N.J., Farr A.L., Randall R.J. Protein measurement with the Folin phenol reagent. J Biol Chem (1951) 193:265–275.[Free Full Text]
  21. Nakamura T., Takeshita I., Inamura T., Tashima T., Fukui V. Expression of the B-chain of plate-let derived growth factor and proliferation activity of human brain tumors. Neurol Med Chir (Tokyo) (1993) 33:205–211.[Medline]
  22. Laemmli U.J. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (1970) 227:680–685.[CrossRef][Medline]
  23. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheet: procedure and some applications. Proc Natl Acad Sci USA (1979) 76:4350–4354.[Abstract/Free Full Text]
  24. Moses H.L., Coffey R.J. Jr., Leof E.B., Lyon R.M., Keski-Oja J. Transforming growth factor β regulation of cell proliferation. J Cell Physiol (1987) 5(Suppl.):1–7.[Medline]
  25. Majack R.A. Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell culture. J Cell Biol (1987) 105:465–471.[Abstract/Free Full Text]
  26. Kubo A., Fukuda N., Soma M., Izumi Y., Kanmatsuse K. Effect of an angiotensin II type 1 receptor antagonist on growth of vascular smooth muscle cells from Wistar-Kyoto rats and spontaneously hypertensive rats. J Cardiovasc Pharmacol (1996) 27:58–63.[CrossRef][Web of Science][Medline]
  27. Fukuda N., Satoh C., Hu W.-Y., et al. Production of angio II by homogeneous cultures of vascular smooth muscle cells from spontaneously hypertensive rats. Arterioscler thromb Vasc biol (1999) 19:1210–1217.[Abstract/Free Full Text]
  28. Satoh C., Fukuda N., Kubo A., et al. Role of endogenous angiotensin II in the expression of growth factors and the cell cycle in vascular smooth muscle cells from SHR. Jpn Heart J (1998) 39:562.
  29. Symons R.H. Small catalytic RNAs. Ann Rev Biochem (1992) 61:641–671.[CrossRef][Web of Science][Medline]
  30. Inoue M. Antisense RNA: its functions and applications in gene regulation-a review. Gene (1988) 72:25–34.[CrossRef][Web of Science][Medline]
  31. Walder R.Y., Walder J.A. Role of RNase H in hybrid-arrested translation by antisense oligonucleotides. Proc Natl Acad Sci USA (1988) 85:5011–5015.[Abstract/Free Full Text]
  32. Xu Z.D., Oey L., Mohan S., et al. Hammerhead ribozyme-mediated cleavage of the human insulin-like growth factor-Il ribonucleic acid in vitro and in prostate cancer cells. Endocrinology (1999) 140:2134–2144.[Abstract/Free Full Text]
  33. Tsuchihashi Z., Khosla M., Herschlag D. Protein enhancement of hammerhead ribozyme catalysis. Science (1993) 262:99–102.[Abstract/Free Full Text]
  34. Menger M., Tuschl T., Eckstein F., Porschke D. Mg2+-dependent conformational changes in the hammerhead ribozyme. Biochemistry (1996) 35:14710–14716.[CrossRef][Web of Science][Medline]
  35. Hendry P., McCall M.J. A comparison of the in vitro activity of DNA-armed and all-RNA hammerhead ribozymes. Nucleic Acid Res (1995) 23:3928–3936.[Abstract/Free Full Text]
  36. Heidenreich O., Eckstein F. Hammerhead ribozyme-mediated cleavage of the long terminal repeat RNA of human immunodeficiency virus type 1. J Biol Chem (1992) 267:1904–1909.[Abstract/Free Full Text]
  37. Taylor N.R., Kaplan B.E., Swidersk P., Li H., Rossi J.J. Chimeric DNA–RNA hammerhead ribozymes have enhanced in vitro catalytic efficiency and increased stability in vivo. Nucleic Acid Res. (1992) 20:4559–4564.[Abstract/Free Full Text]
  38. Shimayama T., Nishikawa F., Nishikawa S., Taira K. Nuclease-resistant chimeric ribozymes containing deoxyribonucleotides and phosphorothioate linkages. Nucleic Acid Res (1993) 21:2605–2611.[Abstract/Free Full Text]
  39. Wellstein A., Zugmaler G., Califano J., et al. Tumor growth dependent on Kaposi's sarcoma derived fibroblast growth factor inhibited by pentosan polysulfate. J Natl Cancer Inst (1991) 83:716–720.[Abstract/Free Full Text]
  40. Perez J.R., Li Y., Stein C.A., et al. Sequence- independent induction of Spi transcription factor activity by phosphoro thioate oligodeoxynucleotides. Proc Natl Acad Sci USA (1994) 91:5957–5961.[Abstract/Free Full Text]
  41. Wilcox J.N. Molecular biology: insight into the causes and prevention of restenosis after arterial intervention. Am J Cardiol (1993) 72:65E–88E.[CrossRef][Medline]
  42. Isaka Y., Akagi Y., Ando Y., et al. Gene therapy by transforming growth factor-β receptor-IgG Fc chimera suppressed extracellular matrix accumulation in experimental glomerulonephritis. Kidney Int (1999) 55:465–475.[CrossRef][Web of Science][Medline]

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