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(Circulation Research. 1995;76:505-513.)
© 1995 American Heart Association, Inc.

Articles

Effects of Antisense c-myb Oligonucleotides on Vascular Smooth Muscle Cell Proliferation and Response to Vessel Wall Injury

Augusto E. Villa, Luis A. Guzman, Earl J. Poptic, Vinod Labhasetwar, Stanley D'Souza, Catherine L. Farrell, Edward F. Plow, Robert J. Levy, Paul E. DiCorleto, Eric J. Topol

From the Center for Thrombosis and Vascular Biology, Department of Cardiology and the Department of Cell Biology, The Cleveland (Ohio) Clinic Foundation (A.E.V., L.A.G., E.J.P., S.D., E.F.P., P.E.D., E.J.T.); the Division of Pediatric Cardiology, University of Michigan Medical School, Ann Arbor (V.L., R.J.L.); and Amgen, Thousand Oaks, Calif (C.L.F.).

Correspondence to Eric J. Topol, MD, The Cleveland Clinic Foundation, 9500 Euclid Ave, Cleveland, OH 44195. E-mail topole@ccsmtp.ccf.org.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Abstract The process of restenosis after arterial balloon dilatation has been demonstrated to involve smooth muscle cell hyperplasia. Initial reports with antisense oligonucleotides directed against the proto-oncogene c-myb suggest marked in vitro specificity and in vivo efficacy. In the present study, we sought to confirm and extend the hypothesis that antisense to c-myb results in a specific antiproliferative effect with a comprehensive assessment by using different oligonucleotide preparations, different species, and tissue and cellular uptake experiments. Phosphorothioate-protected oligonucleotides representing the appropriate sequence for antisense to c-myb and multiple controls were used to inhibit proliferation of platelet-derived growth factor– and fetal bovine serum–stimulated rat, dog, and human aortic smooth muscle cells in vitro and neointimal proliferation in the rat carotid injury model. In vitro experiments using identical culture conditions in rat, dog, and human aortic smooth muscle cells failed to show specificity as well as consistency in growth inhibitory effects that could be attributed to an antisense mechanism. Proliferation of smooth muscle cell growth in culture was consistently inhibited with oligomers containing a contiguous 4-guanosine residue motif. In vivo, the rat carotid injury neointimal hyperplasia was similar for antisense c-myb (0.095±0.009 mm²) and sense c-myb (0.090±0.009 mm²). Fluorescent-labeled oligonucleotides were present in tissue after local delivery via pluronic gel, and their activity rapidly declined over a 72-hour period. Our findings point to the potential nonspecificity and lack of consistency of the antisense oligonucleotide to c-myb in vitro and in vivo. An alternative nonantisense mechanism for the inhibition of smooth muscle cell proliferation, involving contiguous 4-guanosine residues, is proposed.

Key Words: antisense oligonucleotides • arterial injury • proto-oncogenes • smooth muscle cell proliferation • restenosis

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
Percutaneous balloon angioplasty has become a well-established therapy in the management of coronary artery disease. However, in the last 15 years, little has been achieved in the prevention of restenosis after successful angioplasty,^{1 2 3} with failure to demonstrate a significant reduction in the high incidence (30% to 50%) of restenosis.^{4 5} In experimental models, smooth muscle cell (SMC) proliferation has been shown to be one of the key events responsible for restenosis after balloon angioplasty,^{6 7 8} and the involvement of proto-oncogenes in cell proliferation is well documented.^{9 10 11} One of these, the proto-oncogene c-myb, is a DNA-binding protein that appears to regulate cell growth and differentiation.^{12 13} c-myb message is present in low levels in quiescent SMCs but increases substantially after mitogen stimulation. In vitro studies using antisense c-myb oligonucleotides have shown marked inhibition of SMC proliferation.¹⁴ Other antisense oligonucleotides targeting the mRNA for nonmuscle myosin heavy chain, proliferating-cell nuclear antigen (PCNA),¹⁵ and c-myc^{16 17} have inhibited SMC proliferation as well. An in vivo study in the rat carotid artery injury model using mouse antisense c-myb has confirmed these in vitro findings.¹⁸ In addition, the combination of two different antisense oligonucleotides administered after experimental angioplasty has also been demonstrated to inhibit the arterial intimal hyperplasia response after balloon injury,¹⁹ suggesting that antisense technology may have a potential role in the prevention of restenosis after balloon angioplasty. Few studies have raised the issue of nonspecificity with antisense oligonucleotides,^{20 21} and besides the in vivo study mentioned above, no other in vivo studies have corroborated the potential benefits of c-myb mRNA expression inhibition for the treatment of restenosis.

The purposes of the present study were (1) to investigate in vitro the specificity and efficacy of antisense c-myb oligonucleotide in the inhibition of aortic SMC proliferation and (2) to determine whether local delivery of antisense c-myb oligonucleotides decreases neointimal proliferation after balloon injury in the rat carotid artery model.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Synthesis and Purification of Oligonucleotides
Phosphorothioate-protected oligonucleotides were used to inhibit proliferation of platelet-derived growth factor (PDGF)–and fetal bovine serum (FBS)–stimulated rat, dog, and human aortic SMCs in vitro and neointimal proliferation after vascular injury in vivo. The sequences of the different oligonucleotides used for the present study were as follows: 18-mer mouse antisense c-myb (5'-GTG-TCG-GGG-TCT-CCG-GGC-3'), (position 4-22 of the mouse c-myb sequence), which is a 1-bp mismatch with rat c-myb; 18-mer 2-bp mismatch mouse antisense c-myb (5'-GTG-CCG-GGG-TCT-TCG-GGC-3'); 18-mer mouse sense c-myb (5'-GCC-CGG-AGA-CCC-CGA-CAC-3'); 19-mer mouse antisense to interleukin-1ß (IL-1ß) (5'-CAG-TTG-CCA-TAG-CTG-CTT-C-3'); 19-mer 4-bp mismatch antisense to IL-1ß (5'-CAC-TTG-CGA-TAC-CTG-GTT-C-3'); 18-mer human antisense to the BCR-ABL break points in leukemic cells of patients with chronic myelogenous leukemia (CML) (5'-GCT-TTT-GAA-CTC-TGC-TTA-3'); and 16-mer human antisense to CML (5'-CTG-AAG-GGC-TTC-TTC-C-3') (all provided by Genta). The other oligonucleotides used were as follows: 18-mer rat antisense c-myb (5'-GTG-CCG-GGG-TCT-CCG-GGC-3') (position 4-22 of the rat c-myb sequence) and 18-mer scrambled sequence of human antisense c-myb (5'-GCT-GTG-GGG-CGG-CTC-CTG-3') (provided by Amgen). Genta and Amgen oligonucleotides were synthesized on a Milligen 8800 synthesizer; Beaucage reagent was used to sulfurize the internucleotide linkages. Oligonucleotides were purified by use of high-performance liquid chromatography and shown to be >95% pure by rigorous analytical techniques: capillary electrophoresis, analytical reverse-phase chromatography, and phosphorous nuclear magnetic resonance.

In Vitro Effect of Oligonucleotides in Aortic SMC Proliferation
Rat aortic SMCs were plated at a 1:3 split ratio in 10% FBS and made quiescent 24 hours later by changing the medium to 1% control process serum replacement II (Sigma Chemical Co). Three to 5 days later, the cells were stimulated with PDGF (2 ng/mL). At the time of stimulation, 18-mer mouse antisense c-myb and 2-bp mismatch antisense c-myb oligonucleotides were added at 1.6, 16, and 40 µmol/L. Cell proliferation was determined by measuring [³H]thymidine incorporation into DNA at 18 to 22 hours. Following a similar protocol, a second set of experiments was performed to compare further the specificity of the antiproliferative effects of antisense c-myb with other control oligonucleotides. This time oligonucleotides were added at 20 µmol/L and included 18-mer mouse antisense c-myb, 18-mer sense c-myb, 19-mer mouse antisense and 4-bp mismatch antisense to IL-1ß, and 18- and 16-mer human antisense to CML. In a third set of experiments, antisense c-myb, sense c-myb or F-127 pluronic gel alone, and the combination of antisense and gel or sense and gel were tested for their ability to inhibit the growth of rat aortic SMCs, which was assessed by the measurement of cell number. Specifically, rat aortic SMCs at passages 4 to 7 were plated at a 1:3 split ratio in 12-well plates (Costar) using Ham F12/DMEM (1:1) with 10% FBS (Irvine Scientific). Twenty-four hours later, cells were washed two times with PBS and refed with medium containing 0.5% FBS to make cells quiescent. Four days later, the medium was changed to 10% FBS to stimulate growth. Immediately after the addition of 10% FBS, antisense and sense c-myb (10 µmol/L), F-127 pluronic gel, or the combination of oligonucleotide and gel was added to the cells. Four days after stimulation with 10% FBS, cells were trypsinized, and cell number was determined with a hemocytometer.

To further determine if the lack of specificity encountered in the first three experiments could be related to the specific type of oligonucleotides used, another set of in vitro experiments was performed; this time oligonucleotides from a different source, specific against rat c-myb (Amgen), were used. Also, different cell lines and species were tested to determine whether the inhibitory effects differed. The effect of rat antisense c-myb on proliferation of rat, dog, and human aortic SMCs was tested with a similar protocol as described in the first in vitro experiment. A scrambled oligonucleotide sequence of antisense c-myb was used as a control. Toxicity of the antisense and control oligonucleotides was evaluated by using a modification of the method of Shirhatti and Krishna.²² Briefly, rat aortic SMCs were incubated with medium (DV/F12/0.5% FBS) containing 0.2 mCi/mL [¹⁴C]adenine (58 Ci/mmol, NEN) for 12 to 24 hours. Cells were washed two times with 0.5 mL DV/F12/0.5% FBS to remove unincorporated label and then refed 0.5 mL of the same media. At this time, serum and oligonucleotides were added. After 24 hours, [¹⁴C]adenine release was measured by removing 50 µL of the conditioned medium and counting in a scintillation counter. Cell-associated [¹⁴C]adenine was determined by rinsing the cells two times with 0.5 mL cold 5% trichloroacetic acid, solubilizing with 0.75 mL 0.25N NaOH, and counting a 0.5-mL aliquot.

F-127 Pluronic Polymer Gel Preparation and Controlled Release Study
The polymer used in this study for local delivery of oligonucleotides was F-127 pluronic gel (BASF, Inc). This polymer has the novel property of being soluble at 4°C, while solidifying on contact with tissues at 37°C. Controlled-release gel, in general, was formulated by preparing phosphate buffer of pH 7.0 (sodium phosphate dibasic [0.015 mol/L] and potassium phosphate monobasic [0.05 mol/L]). The pH was adjusted with 5N sodium hydroxide. Phosphate buffer (2.3 mL) was cooled to 4°C in a graduated test tube. Seven hundred fifty milligrams of F-127 pluronic polymer and 3 mg of equivalent oligonucleotide previously cooled were added. The mixture was vortexed gently and kept at 4°C for 24 hours. The volume was completed to 3 mL with phosphate buffer previously cooled to 4°C and mixed with occasional and gentle vortexing for 1 hour.

In vitro release of antisense c-myb oligonucleotide from F-127 pluronic gels was carried out at 37°C in PBS (0.154 mol/L), pH 7.4, under perfect sink conditions.^{23 24} Antisense oligonucleotide was mixed with ³²P end-labeled antisense prepared by the T4 polynucleotide kinase method²⁵ (18 527 dpm/µL) at a 2:1 ratio in a pluronic gel as described earlier. Aliquots of the cold pluronic solution (500 µL) at 4°C were transferred to four borosilicate glass test tubes (12x75 mm), each with an internal diameter of 10 mm, and formed a layer of 9.5 mm thickness at the bottom. The gel was brought to 37°C and then incubated with 2 mL PBS at 37°C with a gentle swirled motion on a rotary shaker. At regular intervals of time, buffer was withdrawn without disturbing the bottom gel layer to measure ³²P activity with a Beckman liquid scintillation counter (model LS-3801, Beckman Instruments, Inc). The in vitro study was continued until the pluronic gel completely disappeared.

In Vivo Tissue Uptake, Localization, and Integrity of Oligonucleotides
Fluorescein isothiocyanate (FITC)–labeled and bromodeoxyuridine (BrdU)–labeled oligonucleotides were used for these experiments. BrdU was conjugated for the four–common nucleoside phosphoramidite. FITC was conjugated to the oligonucleotides through the 5' hydroxyl by using a fluorescein amidite.

Twelve Sprague-Dawley rats were used for oligonucleotide tissue detection, integrity, and localization. After balloon injury of the entire left common carotid artery with a 2F Fogarty balloon catheter,²⁶ F-127 pluronic gel loaded with 200 µg of labeled oligonucleotides was applied around the left common carotid artery. Six animals were killed 3, 24, and 72 hours after injury for tissue localization. Another six animals were killed at the same time points for determination of oligonucleotide tissue uptake and integrity.

Tissue localization was determined using FITC- and BrdU-labeled oligonucleotides. Briefly, immediately after the animals were killed, the carotid arteries treated with BrdU-labeled oligonucleotides were gently dissected and retrieved free of surrounding tissue. The distal 15-mm treated segments were placed in zinc formalin for 24 hours. After fixation, the arteries were placed in PBS, and the tissue was processed for immunohistochemistry with anti-BrdU antibodies. For better definition of cell structure and oligonucleotide identification, the histological sections were counterstained with hematoxylin and eosin. The left common carotid artery segments treated with FITC oligonucleotides were dissected and placed in 4% paraformaldehyde for 3 hours. Segments were then removed and cryoprotected by placing them in 30% sucrose in 0.1 mol/L phosphate buffer and frozen at -20°C. Frozen sections were visualized under fluorescence microscopy.

Tissue uptake and integrity of oligonucleotides were analyzed as previously described.²⁷ Briefly, the distal 15-mm segment of the left common carotid artery was digested in 0.5 mL of a lysis buffer (200 mmol/L Tris [pH 8.5], 100 mmol/L EDTA, 1% sodium dodecyl sulfate, and 1 mg/mL proteinase K) at 56°C overnight. After digestion, the solution was spun at 12 000g for 10 minutes at 4°C. Fifty-microliter aliquots of the supernatant were analyzed in a fluorescence spectrometer (Perkin-Elmer) for FITC activity. A standard curve was created with serial dilutions of FITC oligonucleotides in the same lysis buffer. The remainder of the supernatant was diluted to 2.5 mL and ultrafiltrated through a 3-kD cutoff microconcentrator (Amicon) to a final volume of 10 µL. Forty microliters of loading buffer (8 mol/L urea and 1x TBE containing [mol/L] Tris base 0.09, boric acid 0.09, and EDTA 0.002) was added to the sample to a final volume of 50 µL. The entire 50-µL solution was loaded into the gel. The retained materials were analyzed for the presence and integrity of the FITC oligonucleotides by gel electrophoresis, and the bands were identified by UV transillumination.

In Vivo Neointimal Proliferation Inhibition by Local Delivery of Oligonucleotides
In four groups of Sprague-Dawley rats weighing 450 to 530 g, 0.2 mL of F-127 pluronic polymer gel was applied circumferentially to the distal 15 mm of the left common carotid artery adventitial surface after deendothelialization of the entire artery with a 2F Fogarty balloon catheter as previously reported.²⁶ Oligonucleotides were suspended in F-127 pluronic gel at 4°C; for these in vivo experiments, the mouse antisense oligonucleotides were used. The two treatment groups received either 20 µg of mouse antisense c-myb or 200 µg of mouse antisense c-myb. Two control groups received either 200 µg of sense c-myb or plain pluronic gel. In a fifth group, 200 µg of antisense c-myb suspended in 0.2 mL of F-127 pluronic gel was administered intraperitoneally after balloon injury to rule out a systemic effect from the local periadventitial administration. All animal experiments were performed according to the animal welfare policy of the American Heart Association and the Cleveland Clinic Foundation, and the experimental protocol was approved by the animal research committee.

Morphometric and Histological Analysis
Three weeks after balloon carotid injury, rats were killed, and pressure-perfusion fixation was performed as previously described.²⁶ The left common carotid arteries were sectioned every 3 mm from the proximal to the distal ends. Three different sections of the left common carotid artery within the distal 15-mm segment were selected for histological analysis. These sections were embedded in paraffin for sectioning, and duplicate slides were stained with hematoxylin and eosin and Lawson's elastic–van Gieson. Morphometric analysis was performed with a computerized digital microscopic planimetry algorithm (BIOQUANT program) by an observer blinded to drug regimen. Cross-sectional areas of media, intima, and lumen were measured.

Statistical Analysis
Differences between means were analyzed by using ANOVA with Scheffé's test for post hoc multiple comparisons or a two-tailed unpaired Student's t test. Statistical significance was defined as P<.05.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
In Vitro Effect of Oligonucleotides in Aortic SMC Proliferation
In the first set of in vitro experiments (Fig 1), there was a very similar dose-related antiproliferative effect in PDGF-stimulated rat aortic SMCs, with both the mouse antisense and 2-bp mismatch c-myb antisense. Significant inhibition of rat aortic SMC proliferation occurred only with higher concentrations (16 and 40 µmol/L) of both antisense oligonucleotides. There was no significant growth inhibition with either antisense oligonucleotide at lower concentrations (1.6 µmol/L). In the second set of experiments (Fig 2), there was a significant reduction in cell growth, as assessed by [³H]thymidine incorporation, when both the mouse antisense and sense c-myb oligonucleotides were used at 20 µmol/L (antisense c-myb, 249±9 cpm; sense c-myb, 594±4 cpm; and PDGF, 1327±38 cpm). However, the degree of inhibition compared with the PDGF-stimulated control cells was significantly greater in the mouse antisense c-myb–treated group than in the sense c-myb–treated group (81% versus 55%, respectively; P<.05). In addition, a 19-mer mouse antisense and 4-bp mismatch antisense oligonucleotide to IL-1ß controls (462±7 and 411±7 cpm, respectively) and an 18- and 16-mer human antisense oligonucleotide to CML (639±17 and 739±22 cpm, respectively) also showed significant inhibition of growth.

View larger version (15K): [in this window] [in a new window]   Figure 1. Bar graph showing a similar dose-related inhibition of rat aortic smooth muscle cell proliferation with antisense (AS) c-myb and 2-bp mismatch (2-mm) AS c-myb, assessed by [3H]thymidine incorporation. Values are mean±SEM. The asterisks denote statistical significance (P<.0001) compared with platelet-derived growth factor (PDGF)–stimulated control culture.

View larger version (15K): [in this window] [in a new window]   Figure 2. Bar graph showing a significant inhibition of rat aortic smooth muscle cell proliferation assessed by [3H]thymidine incorporation, with 20 µmol/L of antisense (AS) and sense c-myb, 19-mer mouse antisense and 4-bp mismatch (4-MM) AS to interleukin-1ß (IL-1ß), and 16- and 18-mer human AS to chronic myelogenous leukemia (CML). Values are mean±SEM. For each oligonucleotide preparation tested, the significant inhibition was compared with platelet-derived growth factor (PDGF)–stimulated control culture (P<.0001 for each difference).

In the third set of experiments (Fig 3), there was a significant inhibition of growth, assessed by cell count with the addition of either F-127 pluronic gel or 10 µmol/L of mouse AS c-myb oligonucleotides and a further reduction with the addition of both (10% FBS, 548±34 cells; F-127 gel, 415±9 cells; mouse antisense c-myb, 297±4 cells; and mouse antisense c-myb+gel, 264±6 cells). Similar results were seen with 10 µmol/L of sense c-myb oligonucleotides and F-127 pluronic gel (sense c-myb, 313±16 cells; sense c-myb+F-127 gel, 290±15 cells).

View larger version (15K): [in this window] [in a new window]   Figure 3. Bar graph showing a significant inhibition of growth of rat aortic smooth muscle cells with the addition of F-127 pluronic polymer gel, 10 µmol/L of antisense (AS) c-myb or sense c-myb, and a further reduction with the combination of pluronic polymer gel and 10 µmol/L of AS c-myb or sense c-myb assessed by cell count. Values are mean±SEM. The asterisks represent the level of statistical significance for each additive compared with 10% fetal bovine serum (FBS)–stimulated control culture (*P<.05, **P<.001).

In the next two sets of experiments (Fig 4, top and middle) using rat and dog aortic SMCs, there was a similar dose-related nonspecific growth-inhibitory effect with the scrambled control oligonucleotide, but no significant growth inhibitory effect was observed with rat antisense c-myb oligonucleotide at 2, 10, and 20 µmol/L. No evidence of a direct cellular toxic effect was seen with either oligo by [¹⁴C]adenine release measurement. When human aortic SMCs were used (Fig 4, bottom), there was a significant dose-related growth-inhibitory effect with both rat antisense c-myb and control scrambled oligonucleotides. Further assessment of viability by [¹⁴C]adenine release showed no evidence of a direct cellular toxic effect.

View larger version (24K): [in this window] [in a new window]   Figure 4. Top, Bar graph showing a dose-related inhibition of rat aortic smooth muscle cell (SMC) proliferation with scrambled-sequence oligonucleotide (SCR) but no significant inhibition with rat antisense c-myb (AS), assessed by [3H]thymidine incorporation. Middle, Bar graph showing a dose-related inhibition of dog aortic SMC proliferation with SCR but no significant inhibition with AS, assessed by [3H]thymidine incorporation. Bottom, Bar graph showing a dose-related inhibition of human aortic SMC proliferation with both AS and SCR, assessed by [3H]thymidine incorporation. No difference was observed in [14C]adenine release on any of the treated groups compared with the untreated 10% fetal bovine serum (FBS)–stimulated human aortic SMCs. Values are mean±SEM.

In Vitro Release of c-myb From F-127 Pluronic Polymer Gel
Erosion of the gel under in vitro conditions resulted in an almost continuous release of incorporated antisense c-myb oligonucleotide, with 90% release occurring in 51 hours and 100% release by 67 hours. The cumulative percent release of antisense is shown in Fig 5.

View larger version (13K): [in this window] [in a new window]   Figure 5. Graph showing cumulative [32P]antisense c-myb release as a percentage of the initial antisense c-myb loading from F-127 pluronic polymer gel over an extended period. Each data point represents the mean of six values. Values are mean±SD.

Oligonucleotide Tissue Uptake, Integrity, and Localization
Fig 6 shows the tissue localization of the oligonucleotides. At 3 hours after injury, there was diffuse and intense evidence of oligonucleotides in the adventitial layer. In the media, the staining was mild and mainly located in the matrix, with few positive stained cells. At 24 hours, there was intense adventitial staining, with a significant number of medial cells showing cytoplasmic and nuclear staining. The tissue fluorescent activity represented 2% to 4% of the total oligonucleotides implanted, with fluorescent activity persisting even 72 hours after injury. Fig 7 demonstrates the pres-ence of the oligonucleotides in tissue as a function of time. Intact oligonucleotides were found at all time points; however, the amount of intact oligonucleotides recovered from the vessel wall appeared to decrease over time.

View larger version (0K): [in this window] [in a new window]   Figure 6. Color photomicrographs of histological sections from left common carotid artery showing the tissue localization of oligonucleotides. Top, Fluorescence microscopy of fluorescein isothiocyanate (FITC)–labeled oligonucleotides 3 hours after vascular injury showing oligonucleotides in the adventitial layer (original magnification x200). Middle, Fluorescence microscopy of FITC-labeled-oligonucleotides 24 hours after vascular injury showing media nuclear localization of oligonucleotides (original magnification x200). Bottom, Bromodeoxyuridine (BrdU)–labeled oligonucleotides 24 hours after vascular injury. The figure shows in brown dots the oligonucleotides localized intracellularly in the perinuclear zone. The section was processed for immunohistochemistry with anti-BrdU antibody and counterstained with hematoxylin and eosin (original magnification x1500).

View larger version (117K): [in this window] [in a new window]   Figure 7. Electrophoretic analysis of the integrity of fluorescein isothiocyanate (FITC) oligonucleotide extracted from carotid artery tissue at different time points. Lanes are as follows: 1, control FITC oligonucleotides; 2 and 3, 3 hours after vascular injury; 4 and 5, 24 hours after vascular injury; 6 and 7, 72 hours after vascular injury; and 8, nontreated carotid artery (negative control). Note that despite loading with a similar amount of fluorescent activity, the amount of intact oligonucleotides decreased over a 3-day period.

Neointimal Proliferation
Representative cross sections for each set of in vivo experiments are shown in Fig 8. Fig 9 shows the cross-sectional areas of neointima and the intima-to-media (I/M) ratio of the left common carotid artery segments 3 weeks after balloon injury. There was no significant difference in the cross-sectional neointimal areas and the I/M ratio in all five groups (respective values are as follows: local antisense c-myb low dose, 0.104±0.013 mm² and 0.96±0.12; local antisense c-myb, 0.095±0.009 mm² and 0.84±0.07; local sense c-myb, 0.090±0.009 mm² and 0.79±0.08; local plain pluronic gel, 0.102±0.007 mm² and 0.86±0.06; and intraperitoneal antisense c-myb, 0.111±0.012 mm² and 0.92±0.07). The media cross-sectional areas were also similar in all five groups (local antisense c-myb low dose, 0.109±0.002 mm²; local antisense c-myb, 0.113±0.005 mm²; local sense c-myb, 0.114±0.004 mm²; local plain pluronic gel, 0.119±0.003 mm²; and intraperitoneal antisense c-myb, 0.120±0.006 mm²).

View larger version (0K): [in this window] [in a new window]   Figure 8. Color photomicrographs of representative histological sections from left common carotid artery segments 3 weeks after balloon injury. Top, Balloon-injured segment covered with placebo polymer. Middle, Balloon-injured segment covered with antisense c-myb. Bottom, Balloon-injured segment covered with sense c-myb. For all panels, Lawson's elastic–van Gieson stain was used (original magnification x100).

View larger version (36K): [in this window] [in a new window]   Figure 9. Bar graphs in the five groups of rats with eight animals that underwent balloon injury, analyzed per group. A, Neointimal proliferation of left common carotid arterial segments covered by F-127 pluronic polymer gel. There was no significant inhibition in any group compared with the placebo pluronic polymer group. B, Intima-to-media (I/M) ratio of arterial segments covered by F-127 pluronic polymer gel. The I/M ratio was not significantly decreased in any group compared with the placebo pluronic polymer group. AS indicates antisense; MYB, c-myb. Values are mean±SEM.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We have shown in the initial in vitro studies that antisense oligonucleotide to c-myb inhibits rat aortic smooth muscle cell proliferation only at higher doses; however, several other oligonucleotides tested did so as well when administered at higher doses. Further experiments corroborated the nonspecific growth-inhibitory effects seen with control oligonucleotides and demonstrated a lack of consistency of growth inhibition by antisense c-myb across different cell lines and species. In vivo studies in the rat carotid artery model failed to demonstrate a significant inhibition of neointimal proliferation 3 weeks after balloon injury by local adventitial delivery of antisense c-myb, in spite of evidence of tissue uptake, nuclear localization, and preservation of integrity of the oligonucleotides. Experiments to demonstrate inhibition of c-myb message expression were not pursued, since the effects of inhibition of cell growth were nonspecific, inconsistent, and, at best, marginal in potency. The present study also extends previous findings by testing a dose-response relation for antisense c-myb in vivo, differentiating a systemic versus local effect (using intraperitoneal injection), and characterizing the release of the antisense oligonucleotides from the local polymer delivery system.¹⁸

In Vitro Experiments
In these experiments, we tried to determine the specificity and efficacy of the antiproliferative effect of antisense oligonucleotides against rat c-myb mRNAs by using as controls 2-bp mismatch antisense c-myb, scrambled sequence of antisense c-myb, sense c-myb, 19-mer mouse antisense and 4-bp mismatch antisense to IL-1ß, and 18- and 16-mer human antisense oligo to the BCR-ABL break points in leukemic cells of patients with CML. In the first set of experiments, mouse antisense c-myb and 2-bp mismatch antisense c-myb at lower doses (1.6 µmol/L) did not significantly inhibit SMC proliferation. At higher concentrations (16, 20, and 40 µmol/L), the control 2-bp mismatch antisense c-myb caused growth inhibition similar to the antisense c-myb. Sense c-myb, 19-mer mouse antisense and 4-bp mismatch antisense to IL-1ß, and 16- and 18-mer human antisense to CML also caused significant growth inhibition, but the effect was less pronounced.

Question of Specificity
Several in vitro studies have shown that the antiproliferative effects of antisense oligonucleotides targeted against c-myb, nonmuscle myosin heavy chain, c-myc, and PCNA are specific.^{14 15 16 17 28} Corroborating this are the findings that (1) the sense oligonucleotide of the respective target had no inhibitory effect at the same doses as the antisense oligo, (2) antisense mismatch sequences of the respective target also had no inhibitory effect, and (3) expression of the respective target mRNA was diminished in cells treated with antisense but not in those treated with sense or mismatch antisense oligos.

However, our findings suggest that nonspecific effects were involved in the SMC growth inhibition observed with antisense c-myb and the control oligonucleotides. Yaswen et al²⁹ have also previously demonstrated a nonantisense antiproliferative effect of phosphorothiate oligonucleotides with a 4-guanosine (4-G) sequence in epithelial cells. Recent work by Burgess et al³⁰ indicates that antisense c-myb oligonucleotide sequences that contain a 4-G residue motif are predictably growth inhibitory in rabbit SMCs in vitro and ex vivo. Of note, the rat and rabbit c-myb oligonucleotide sequences are identical. The degree of inhibition observed in their studies depended on the context sequence within which the 4-G motif was contained, with the scrambled 4-G sequence being the most inhibitory of the three 4-G oligonucleotides used in our studies. The experiments reported here confirm and extend the observations of Burgess et al to include the inhibitory effect of the 4-G sequences on dog and human SMCs. The nonspecific binding of oligonucleotides to essential components (proteins) of cells has been referred to as an aptamer effect.^{31 32 33}

Other nonspecific nonantisense mechanisms may include the following: (1) the charged oligonucleotide polyanion properties mimicking heparin by binding growth factors,³³ (2) nonspecific cellular activation of the SPI transcription factor, (3) formation of double-stranded RNA, which induces -interferon, a potent inhibitor of SMC proliferation,^{34 35} and (4) the induction of the double-stranded RNA-regulated elF-2 kinase. This kinase prevents the formation of the initiation complex necessary for translation, thus inhibiting protein synthesis and cell proliferation.^{36 37} Of these possibilities, we suspect that the significant inhibition observed with the antisense, mismatch, or scrambled oligonucleotides could be secondary to an aptamer-inhibitory effect. Of note, each of these oligonucleotides achieving SMC inhibition in our experiments contained a sequence with four contiguous guanosine (G) residues. The potential aptamer effect of the 4-G sequence is further exemplified in the other published reports in which phosphorothiate oligonucleotides were administered without a viral vector.^{18 28 38 39} In each of these studies, only the antisense sequence contained the 4-G residues.

Lack of Consistency
Another important observation in the present study was the lack of consistency observed with antisense technology. Antisense c-myb oligonucleotides from two different commercial sources had disparate inhibitory effects on rat aortic SMCs. The same antisense c-myb also had inconsistent inhibitory effects on different cell species. Woolf et al²⁰ showed in studies in vivo that antisense oligonucleotides do not need to be perfectly complementary to their RNA target sequences for cleavage to occur. However, perfectly matched oligonucleotides were more effective than partial matches in cleaving their target RNAs.²⁰ Although the extent of complementarity to mRNA will thus vary for different species,²⁰ the severity of inconsistency with rat antisense directed to rat smooth muscle cells was particularly surprising. Substantial inconsistency has also been observed in the literature (Table).^{18 19 38 39 40 41 42} Morishita et al¹⁹ reported that PCNA antisense oligonucleotide alone did not inhibit SMC proliferation. In contrast, other investigators observed in vitro and in vivo a significant inhibitory effect with PCNA antisense oligonucleotides.^{15 40}

View this table: [in this window] [in a new window]   Table 1. In Vivo Studies of Antisense Oligonucleotides to Inhibit the Response to Vascular Injury

Other inconsistencies in the literature are conspicuous and noteworthy (Table). Although Morishita et al⁴¹ were unable to achieve significant in vivo SMC inhibition without a liposome and viral vector delivery system, other groups with the same oligonucleotide target have been successful without such a delivery system.^{19 38 39 40 42} Further, antisense directed to PCNA in the same experimental model had little inhibition effect in one group of experiments¹⁹ but substantial inhibition by others.⁴⁰ Using the same proto-oncogene cyclin targets, two groups have reported differences in the extent of inhibition.^{19 41 42}

Lack of In Vivo Effect
The lack of a significant neointimal proliferation inhibition after balloon vascular injury in the c-myb–treated group is in disagreement with results recently published by another group of investigators despite the use of exactly the same 18-mer mouse antisense c-myb.¹⁸ One explanation for this difference may be that the rats used in the present study were bigger (450 to 530 g) and therefore probably older. Recent data suggest that SMCs from senescent rats proliferate more actively after injury than SMCs from young rats^{43 44} and possibly are less sensitive to antiproliferative agents. However, we have recently reported the lack of an in vivo antiproliferative effect of antisense to rat c-myb when using young animals.⁴⁵ Another difference in our experiments, although less likely to explain the negative results, was that rats were killed at 3 weeks after balloon vascular injury rather than at 2 weeks. A "catch-up" or rebound phenomenon after an undetected initial short growth inhibition period cannot be completely excluded in the present study. However, if that is the case, such a response will minimize the importance of using antisense c-myb as a potential therapeutic strategy.

In the present study, we have demonstrated tissue uptake with nuclear localization and the integrity of the oligonucleotides from our periadventitial delivery. The release kinetics of the c-myb antisense from F-127 pluronic gel revealed a rapid delivery with completion by 67 hours, approximating first-order kinetics. Via fluorescent labeling, we demonstrated the diminutive presence of oligonucleotides at 72 hours. The small amounts of oligonucleotides present transiently in the arterial wall may not be sufficient to inhibit cell proliferation. In contrast, Morishita et al⁴¹ reported a significant antiproliferative effect of cdk 2 kinase antisense oligonucleotide only when it was delivered by use of enhanced hemagglutinating virus of Japan (HVJ) liposome–mediated transfer but not with direct transfer. In their study, using fluorescence, oligonucleotides were demonstrated at high concentration even 2 weeks after transfection.⁴¹ Accordingly, we hypothesize that the absence of an in vivo effect is related to suboptimal transfection, with respect to both the efficiency and durability of the effect.

Conclusion
The results in the present study exemplify the problems that may be encountered at the present time with antisense technology. Our in vitro and in vivo studies have shown that antisense therapy may not be as specific or consistent as other studies have previously suggested. It is critical that specificity and consistency be established in cell culture experiments and that the results elicited in vivo be concordant with such experiments in order to accept cell biology studies as a meaningful surrogate.^{46 47 48 49} In vivo studies combining antisense c-myb with another antisense oligonucleotide targeted to a different proto-oncogene, the use of better continuous drug release systems with more sustained release properties, enhanced methods of ensuring a high level of intracellular uptake, and studies in larger animal models with single or combined targets are needed. The precise mechanism of the antisense effect, particularly when there is lack of a viral vector to facilitate cellular uptake, needs to be resolved to determine whether an aptamer or true antisense effect is operative. Our findings, combined with previous reports, suggest that the presence of four contiguous guanosine residues may be associated with an aptamer effect, which can be differentiated from a hybridization-dependent antisense mechanism. Further investigation is clearly warranted before applying this novel technology for the prevention of postangioplasty restenosis in humans.

	Acknowledgments

 
The authors thank Genta, Inc, and Amgen, Inc, for synthesizing all of the oligonucleotides used in this study. This work was supported by National Institutes of Health grant HL-29582 to Dr DiCorleto. The efforts of Drs Labhasetwar and Levy were supported by American Heart Association Grant-in-Aid 94-1538. We also thank Farhad Faroudi for technical assistance.

Received October 24, 1994; accepted December 21, 1994.

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