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(Circulation. 1997;95:669-676.)
© 1997 American Heart Association, Inc.

Articles

Antisense Oligonucleotide Inhibition of PDGFR-ß Receptor Subunit Expression Directs Suppression of Intimal Thickening

Martin G. Sirois, PhD; Michael Simons, MD; Elazer R. Edelman, MD, PhD

Harvard-MIT Division of Health Sciences and Technology, Cambridge (M.G.S., E.R.E.); Brigham and Women's Hospital (E.R.E.), Boston; and Beth Israel Hospital Departments of Medicine, Harvard Medical School (M.S.), Boston, Mass.

Correspondence to Martin G. Sirois, PhD, Division of Health Sciences and Technology, Massachusetts Institute of Technology, 20A-108, 18 Vassar St, Cambridge, MA 02139. E-mail mgsirois@mit.edu.

	Abstract

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Background The elucidation of molecular mechanisms of vascular cell biology has markedly influenced our thinking on the pathophysiology of vascular disease. Antisense oligonucleotide gene therapy has helped identify proteins critical to cell-cycle progression and proliferation and possible therapeutic strategies to combat human disease. This approach, however, has not yet been used to examine the contribution of chemotactic proteins and/or their receptors. Platelet-derived growth factor-BB (PDGF-BB) released from activated platelets adherent to subendothelial connective tissue is a principal smooth muscle cell chemotactic factor.

Methods and Results A series of experiments was performed to assess the capacity of antisense oligonucleotides to reduce PDGF-ß receptor subunit (PDGFR-ß) expression and the contribution of PDGFR-ß in neointimal formation. Sustained, direct, and local perivascular administration of two different antisense oligonucleotide sequences complementary to PDGFR-ß mRNA almost completely abolished the expression of PDGFR-ß protein in the intima and media of injured carotid arteries and decreased neointimal formation by 80% and 60%, respectively. Furthermore, neointimal formation correlated precisely with PDGFR-ß expression in an exponential fashion.

Conclusions Thus, myointimal proliferation depends on both PDGFR-ß overexpression and its activation by PDGF-BB. Removal of either of these two elements can suppress neointimal formation.

Key Words: angioplasty • growth substances • restenosis • muscle, smooth

	Introduction

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It is now widely accepted that within the first 2 days of vascular injury, damaged and dying medial vSMCs release growth promoters such as bFGF. This induces vSMC proliferation for the next 3 to 5 days, delineating the first wave of the vascular healing process.^{1 2 3} The second and third waves rely on migration of medial vSMCs and their proliferation within the neointima.⁴ It is thought that half of the migrating vSMCs will undergo three rounds of cell-cycle proliferation in the intima, ultimately representing 90% of the final cell count in the neointima. The other half of the migrating vSMCs do not divide and account for the remaining 10% of the intimal cell count.¹ vSMCs are observed within the neointima as soon as 3 days after the injury. Their number peaks within 2 weeks of injury and remains relatively constant for up to 1 year.⁵ Several molecules such as angiotensin II, transforming growth factor-ß, bFGF, and PDGF-BB might act as vSMC chemotactic factors during the second wave of cellular events.⁴ PDGF-BB has received particular attention because it is both mitogenic for cultured vSMCs through activation of either PDGF receptor (PDGFR- or PDGFR-ßß) and chemotactic through the activation of PDGFR-ßß.⁶ In vivo, however, PDGF-BB acts predominantly as a chemotactic factor on vSMCs. Injection of this growth factor increased vSMC migration by 10- to 20-fold but proliferation by no more than 2-fold,⁷ and polyclonal anti-PDGF antibodies blocked the migration of vSMC migration but not their proliferation.⁸ It is therefore reasonable to postulate that PDGF-BB plays a critical role in intimal thickening during the first 2 weeks after a vascular lesion is sustained.

PDGFR-ß is specifically expressed in mesenchymal cells such as vSMCs and fibroblasts.⁹ Basal expression in the medial vSMCs of the normal artery increases within days of injury.¹⁰ What is not known is whether PDGF receptor expression is directly related to the extent of neointimal hyperplasia. Antisense oligonucleotide gene therapy enables us to examine this question.^{11 12 13 14 15 16 17 18} Antisense oligonucleotide sequences hybridize^{19 20 21} with targeted mRNA or gene regions at ribosomal or nuclear sites, preventing mRNA translation into protein.²² To date, antisense oligonucleotides directed against growth-regulatory or cell-cycle genes (c-myb, c-myc, PCNA, cdc2, and cdk2) involved in vSMC proliferation after injury have successfully altered intimal hyperplasia.^{11 12 13 14 15 16 17 18} Yet to the best of our knowledge, no one has used antisense sequences to prevent the expression of chemotactic proteins or their receptors. We examined these issues by examining the effect of antisense phosphorothioate-oligodeoxyribonucleotide sequences complementary to PDGFR-ß mRNA on PDGFR-ß protein expression and intimal thickening after vascular injury. The sustained release of PDGFR-ß mRNA antisense oligonucleotide reduced PDGFR-ß protein expression and intimal thickening in injured rat carotid arteries in an exponentially correlative fashion.

	Methods

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Induction of Intimal Hyperplasia
Balloon denudation of common carotid arterial endothelium was performed in male Sprague-Dawley rats (350 to 425 g; Charles River Breeding Laboratories, Kingston, Mass). The rats were anesthetized with injections of ketamine HCl 75 mg/kg IP (Ketaset, Aveco Co) and xylazine HCl 5 mg/kg IP (Rompun, Miles Inc). After exposure of the left common external carotid artery, a 2F Fogarty balloon catheter (American Edwards Laboratories) was inserted through an arteriotomy into the common carotid artery to the aortic arch, insufflated sufficiently with air to produce slight resistance, and withdrawn three times. On removal of the catheter, the external carotid artery was ligated, the wounds were closed, and the rats were returned to their cages. Rats were killed at different periods of time (7, 14, and 28 days) after injury with an overdose of ketamine and xylazine, exsanguinated, and perfused with 50 mL Ringer's lactate solution. The treated segment of the common carotid artery was removed, cut into two equal segments, and fixed in 5% formalin solution. The segments were embedded in paraffin, and eight 6-µm sections were obtained by microtome along the length of the specimen. Sections were stained with hematoxylin and eosin, and the areas of the intima, media, and adventitia; the intima-to-media-area ratio; and the percent of luminal occlusion were calculated for each arterial segment by use of computerized digital planimetry with a dedicated video microscope and customized software. The nature of specimen treatment was kept from investigators until after completion of the data analysis.

Antisense Oligonucleotides Therapy
To study the possible contribution of PDGFR-ß to neointimal formation, antisense oligonucleotide sequences to the receptor subunit were applied directly to balloon catheter–denuded carotid arteries. We used two different antisense oligonucleotide phosphorothioate backbone sequences to the murine PDGFR-ß mRNA (antisense 1, AS1–PDGFR-ß: TAT CAC TCC TGG AAG CCC, nucleotides 4 through 21; antisense 2, AS2–PDGFR-ß: TCT GAG CAC TAA AGC TGG, nucleotides 22 through 39). Neither sequence contained more than two consecutive guanosines. Two scrambled phosphorothioate sequences (scramble 1, SCR1 GTG ATA GTA TGC CGA GCA; scramble 2, SCR2 CGT TAC GTA AGC CTA GGA) were used as controls. All sequences were synthesized at the Massachusetts Institute of Technology Biopolymers Laboratory. The oligonucleotides were deprotected, dried down, resuspended in Tris-EDTA (10 mmol Tris, pH 7.4, and 1 mmol EDTA, pH 8.0), and quantified by spectrophotometry. To sustain the release and ensure the local administration of the oligonucleotide sequences directly to the injured arteries, the oligomers were embedded within EVAc (DuPont Co) matrix release devices as previously described.^{18 23 24 25} After the endothelial denudation of the left common carotid artery, the EVAc devices containing 400 µg of the scrambled or antisense PDGFR-ß oligomers were placed adjacent to the injured carotid arteries. In 14 days, 65% of the compound was released with zero-order kinetics, and it has been estimated that 1% of the released oligomer would be delivered to the blood vessel wall from these types of devices.^{18 24}

Immunohistochemistry of PDGFR- and PDGFR-ß Expression
Expression of PDGFR- and PDGFR-ß was determined immunohistochemically. Arterial sections were deparaffinized in xylene and ethanol baths; endogenous peroxidase activity was quenched in a solution of methanol (200 mL) plus hydrogen peroxide (3%; 50 mL); and nonspecific binding antibody binding was prevented by preincubation of the tissues with serum (1:10) from species other than those used to raise the primary antibody. Arterial sections were then exposed to the primary antibody, PDGFR- IgG (Santa Cruz Biotechnology, Inc), or rabbit polyclonal anti-human PDGFR-ß IgG (UBI) diluted (1:100, 1:200, 1:500, or 1:1000) or rinsed with PBS, and incubated with a biotinylated goat anti-rabbit IgG (1:400; Dako). Dot blot and Western blot analyses were performed to confirm the cross-reactivity of both rabbit antibodies to rat proteins. Peroxidase labeling was achieved with an incubation by use of avidin/peroxidase complex (Vector Labs Inc), and antibody visualization was established after a 5-minute exposure to 0.05% 3,3'-diaminobenzidine (Sigma Chemical Co) in 0.05 mol/L Tris-HCl, pH 7.6, with 0.003% hydrogen peroxide. The arteries were counterstained by rapid immersion (10 seconds) in Gill's hematoxylin No. 3 solution and rinsed in tap and distilled water.

Cell Culture
vSMCs of rat thoracic aorta were isolated by the explant technique.²⁶ The cells were seeded in culture dishes (35 mm); grown to confluence in DMEM supplemented with 10% FBS (complement-heat inactivated), penicillin (50 U/mL), and streptomycin (50 µg/mL); and used between the 6th and 10th passages. At confluence, the medium was replaced with DMEM, 0.1% FBS, and antibiotics; two groups of cells were treated with either AS1–PDGFR-ß or SCR1–PDGFR-ß (direct application not embedded into EVAc matrices) at 0, 24, and 48 hours, whereas a third group was untreated and served as control. PDGF-BB (10 ng/mL) was added, and total proteins from the cells were collected 0, 1, 3, 6, 12, 24, and 48 hours later.

Western Blot Analysis of PDGFR- and PDGFR-ß Protein
Total proteins were prepared by washing the cells with ice-cold PBS, and the addition of 100 µL of Laemmli buffer containing EDTA 1 mmol/L, phenylmethylsulfonyl fluoride 1 mmol/L, leupeptin 10 µg/mL, and NaVO₃ 1 mmol/L. The extracted cell proteins were boiled for 5 minutes, and a 30-µL aliquot (30 µg protein) of each sample was separated by 7.5% SDS-PAGE under reducing conditions (Minigel Apparatus, Bio-Rad) and transblotted onto 0.45-µm polyvinylidene difluoride membranes (Millipore). The membranes were blocked in TBS–5% Blotto (Tris-HCl 10 mmol/L, NaCl 150 mmol/L, pH 7.85; 5% nonfat dry milk; Bio-Rad) for 1 hour at room temperature with gentle agitation. Membranes were washed with 0.05% TBS and Tween 20–TBS (Bio-Rad) and incubated with rabbit polyclonal anti-human PDGFR-ß IgG antibodies (dilution, 1:200 in Tween 20–TBS) for 2 hours at room temperature. The membranes were washed with Tween 20–TBS and incubated with alkaline-phosphatase goat anti-rabbit IgG (1:100) for 2 hours at room temperature. Membranes were washed with Tween 20–TBS and TBS, and alkaline phosphatase bound to secondary antibodies was revealed by chemiluminescence (Bio-Rad kit). Prestained molecular weight marker proteins (Bio-Rad) were used as standards for SDS-PAGE. To probe the immunoblots with second antiserum, the polyvinylidene difluoride membranes were stripped by incubation in 62.5 mmol/L Tris-HCl, pH 6.7, 2% SDS, and 100 mmol/L 2-mercaptoethanol for 30 minutes at 50°C with gentle agitation. The blots were then washed twice with TBS and then washed at least five times to remove traces of 2-mercaptoethanol. Then the blots were incubated with polyclonal anti-human PDGFR- antibodies (dilution, 1:200 in Tween 20–TBS) and processed as described above.

Statistical Analysis
Data are mean±SEM. Statistical comparisons were determined by ANOVA followed by an unpaired Student's t test with Bonferroni's correction for multiple comparisons. Data were considered to be significantly different if P<.05 was observed.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Neointimal Hyperplasia: Effects of PDGFR-ß mRNA Antisense Oligonucleotides
Neointimal formation determined 14 days after balloon deendothelialization of rat common carotid arteries served as controls for all subsequent experiments. At this time, an intima-to-media-area ratio of 1.37±0.15 was observed (Fig 1). Antisense sequences directed against the PDGFR-ß mRNA were used to reduce receptor subunit expression. The sustained release of antisense oligonucleotide sequences AS1–PDGFR-ß or AS2–PDGFR-ß from EVAc matrices placed adjacent to the injured artery reduced the intima-to-media-area ratio to 0.27±0.09 and 0.55±0.11, but neither scrambled oligonucleotide sequence significantly affected neointimal thickening (SCR1, 1.5±0.12; SCR2, 1.66±0.13; Fig 1). Medial areas were no different in any treated or control groups (data not shown).

View larger version (48K): [in this window] [in a new window]   Figure 1. Effects of mRNA PDGFR-ß antisense oligonucleotides on neointimal formation. After balloon-denuding carotid arterial injury, antisense oligonucleotide sequences corresponding to fragments 4 through 21 (AS1) or 22 through 39 (AS2) or scrambled oligonucleotides of fragments 4 through 21 (SCR1) or 22 through 39 (SCR2) of the 5' region of PDGFR-ß mRNA were released into the perivascular space of injured vessels from implanted EVAc matrices. The rats were killed 14 days later, and the extent of neointimal hyperplasia was expressed as the mean intima-to-media-area ratio±SE from five to six rats per group. *P<.001 vs normal rats subject to balloon injury (BI).

Protein Expression of PDGFR- and PDGFR-ß
In the absence of vascular injury, basal expression of PDGFR-ß was observed on medial vSMCs. We identified 26.5±2.5% of these cells immunohistochemically with an antibody that specifically recognizes the PDGFR-ß protein (Figs 2 and 3a). Fourteen days after a denuding injury, PDGFR-ß protein doubled on medial vSMCs (51.2±5%, P<.001) and became evident on 74.5±2.5% of the intimal cells (Figs 2 and 3b). The sustained perivascular release of both antisense sequences significantly reduced PDGFR-ß expression in both vascular compartments, yet the sequence closer to the 5' mRNA end, AS1–PDGFR-ß, was more potent at reducing receptor subunit and neointimal formation. Two weeks after the treatment of vascular injured carotid arteries with AS1–PDGFR-ß, only 4.4±1.8% of medial cells and 2.8±1.6% of intimal cells retained PDGFR-ß expression (P<.001 versus controls; Figs 2 and 3c). The AS2–PDGFR-ß oligonucleotide reduced these values to 15.9±5.2% and 19.1±5.2%, respectively (P<.001 vs controls; Figs 2 and 3d). Scrambled oligonucleotide sequences had no effect on receptor subunit expression (data not shown). The suppression of neointima with application of antisense PDGFR-ß oligomers followed inhibition of PDGFR-ß expression in an exponential fashion [intima-to-media-area ratio=e^(ß/)], where ß is the percent of all cells expressing the PDGFR-ß and is defined as the exponential constant. Intimal thickening correlated with medial PDGFR-ß expression with a of 17.64 (P<.01, r=.82; Fig 4A) and with intimal receptor expression with a of 0.32 (P<.001, r=.96; Fig 4B).

View larger version (25K): [in this window] [in a new window]   Figure 2. Quantitative assessment of antisense oligonucleotide regulation of PDGFR-ß expression in injured carotid arteries. In the absence of injury, baseline expression of PDGFR-ß reached 26.5±2.5% of all medial cells. Balloon-denuding injury (BI) led to overexpression of PDGFR-ß in both the media (black bars) and neointima (stippled bars). Both antisense oligonucleotide sequences (AS1 and AS2) to PDGFR-ß mRNA reduced the PDGFR-ß expression 14 days after the vascular injury. *P<.05 and ***P<.001 vs noninjured rats; P<.001 vs normal rats subjected to balloon arterial denudation.

View larger version (84K): [in this window] [in a new window]   Figure 3. Antisense oligonucleotide regulation of PDGFR-ß expression on representative cross sections of injured carotid arteries. In the absence of injury (a), baseline expression of PDGFR-ß reached 26.5±2.5% of all medial cells. Balloon-denuding injury led to overexpression in both the media and neointima (b). Both antisense oligonucleotide sequences complementary to PDGFR-ß mRNA reduced receptor subunit expression 14 days after the vascular injury (c, d). Magnification x400.

View larger version (12K): [in this window] [in a new window]   Figure 4. Correlation of antisense regulation of PDGFR-ß expression and neointimal hyperplasia in injured carotid arteries. Shown is the expression of PDGFR-ß in the media (A) and the intima (B) vs the intima-to-media-area ratio 14 days after balloon carotid arterial injury. Data were obtained from rats subject to balloon injury (BI) but not to antisense oligonucleotide sequences treatment () and from rats that were treated either with AS1–PDGFR-ß () or AS2–PDGFR-ß (). Exponential fits were obtained in both cases.

Specificity of the antisense oligonucleotide effect for PDGFR-ß mRNA was demonstrated through similar immunohistochemical identification of PDGFR- protein expression. In the absence of vascular injury, PDGFR- expression was observed on 32.8±4.6% of medial vSMCs (Figs 5a and 6). Fourteen days after denuding injury, PDGFR- expression increased on medial vSMCs (52.7±3.4%, P<.001) and was noted on 57.3±4.2% of the intimal cells (Figs 5b and 6). Despite their effects on PDGFR-ß expression, the sustained perivascular release of either antisense sequences for 14 days after a vascular injury did not affect the PDGFR- expression. PDGFR- protein expression in the media and intima of rat carotid treated with AS1–PDGFR-ß was 58.5±3.2% and 61.5±2.8%, respectively (Figs 5c and 6) and 59.4±3.5% and 62.9±3.8%, respectively, for rats treated with AS2–PDGFR-ß (Figs 5d and 6). Treatment with scrambled oligonucleotide sequences did not alter the expression of PDGFR- compared with control rats (data not shown).

View larger version (80K): [in this window] [in a new window]   Figure 5. Antisense oligonucleotide regulation of PDGFR- expression on representative cross sections of injured carotid arteries. In the absence of injury (a), baseline expression of PDGFR- reached 32.8±4.6% of all medial cells. Balloon-denuding injury led to overexpression of PDGFR- in both the media and neointima (b). Neither of the antisense oligonucleotide sequences complementary to the PDGFR-ß mRNA reduced PDGFR- expression 14 days after the vascular injury (c, d). Magnification x400.

View larger version (36K): [in this window] [in a new window]   Figure 6. Quantitative assessment of antisense oligonucleotide regulation of PDGFR- expression in injured carotid arteries. In the absence of injury, baseline expression of PDGFR- reached 32.8±4.6% of all medial cells. Balloon-denuding injury (BI) led to overexpression of PDGFR- in both the media (black bars) and neointima (dotted bars). Both antisense oligonucleotide sequences (AS1 and AS2) to PDGFR-ß mRNA did not reduce the PDGFR- expression 14 days after the vascular injury. ***P<.001 vs noninjured rats.

Protein Expression of PDGFR-ß on Cultured vSMCs
vSMCs were grown to confluence on 35-mm Petri dishes and then kept quiescent in DMEM with 0.1% FBS; AS1–PDGFR-ß (20 µmol/L) or SCR1–PDGFR-ß oligonucleotide (20 µmol/L) was added at 0, 24, and 48 hours. A third group of cells was untreated with oligonucleotide and served as control. Two days after the first oligonucleotide application, PDGF-BB (10 ng/mL) was added in each group. At 0, 1, 3, 6, 12, 24, and 48 hours after the addition of PDGF-BB, the cells were washed with cold PBS, Laemmli buffer (100 µL) was added, total proteins were collected and quantified by bioassay, and the expression of PDGFR-ß at each time point was determined by Western blot electrophoresis and quantified by image densitometry. Significant baseline PDGFR-ß protein expression was noted in vSMCs (Fig 7). These values decreased by 53% 1 hour after stimulation with PDGF-BB and by an additional 32% 11 hours after that, to be reexpressed near baseline levels 48 hours after initial stimulation. AS1–PDGFR-ß suppressed protein expression by >75% at baseline and for the duration of the experiment (Fig 7). These effects were specific for the PDGFR-ß target gene because PDGFR- protein expression was unaffected by the antisense PDGFR-ß oligonucleotide sequence. The SCR1–PDGFR-ß oligonucleotide sequence had no effect on the normal pattern of PDGFR-ß protein expression seen in control vSMCs at baseline and after stimulation with PDGF-BB (data not shown).

View larger version (29K): [in this window] [in a new window]   Figure 7. Antisense regulation of PDGFR-ß and PDGFR- expression on cultured vSMCs. Quiescent confluent rat vSMCs were stimulated with 10 ng/mL PDGF-BB, and total proteins were collected in Laemmli buffer 0, 1, 3, 6, 12, 24, and 48 hours later. One group of control cells was left without additional therapy (), whereas an identical cohort was treated with 20 µmol/L AS1–PDGFR-ß oligonucleotide 48 and 24 hours and immediately before PDGF-BB exposure (). Total protein (30 µg per lane) was applied on SDS-PAGE under reducing conditions. PDGFR-ß and PDGFR- protein expression was revealed by Western blot electrophoresis and immunohistochemistry and quantified by image densitometry.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In previous reports, we and other investigators showed that antisense oligonucleotide sequences complementary to DNA binding proteins and cell-cycle regulator genes such as c-myb, c-myc, cdc2, cdk2, nonmuscle myosin, and PCNA inhibited target protein expression, suppressed vSMC proliferation in vitro and in vivo, and inhibited neointimal formation in injured arteries of different animal species.^{11 12 13 14 15 16 17 18 27 28 29 30} To date, targeted genes were principally those involved in cell-cycle progression. However, these genes are not unique to vSMCs but also are expressed in other cell types, and their use might induce side effects in tissues with high rates of proliferation. Growth factors play a central role in all phases of the vascular response to injury, yet no studies have reported on the consequences of antisense sequences directed against growth factors and/or their receptors. PDGF, for example, is critical to vSMC migration and intimal thickening^{1 7 31} in a manner fairly selective for vSMCs^{7 8 31} and as a result became the focus of the present manuscript.

We used two antisense oligonucleotide sequences selective for either positions 4 through 21 or 22 through 39 of the PDGFR-ß mRNA. Because PDGFR-ß expression is reexpressed after initial downregulation following PDGF-BB stimulation in vitro (Fig 7) and is manifest over the full 2-week period after in vivo injury (Figs 2 and 3), the oligonucleotides were embedded in EVAc matrices to provide a sustained release during the entire experimental procedure. Previous studies demonstrated the need to match the kinetics of oligonucleotide release to the kinetics of antisense target gene expression. When gene expression is prolonged, as it is for c-myc, a more sustained oligonucleotide release device was required to demonstrate the biological effect.¹⁸ Sustained release of the two antisense oligonucleotide sequences complementary to PDGFR-ß mRNA reduced arterial intimal thickening by 80% and 60%, respectively. In normal rat carotid arteries, 25% of the medial vSMCs stained positive for PDGFR-ß protein. Two weeks after vascular injury, this expression more than doubled in medial vSMCs, and 75% of the cells forming the neointima stained positive. Interestingly, while both antisense sequences reduced PDGFR-ß protein expression below the baseline level (25%) observed in the media of uninjured rat carotid arteries, the oligonucleotide sequence closer to the 5'-mRNA region was almost four times more potent at inhibiting PDGFR-ß expression in medial and intimal vSMCs. The variable response to these two sequences enabled delineation of a correlation between PDGFR-ß levels and neointimal potential. In arteries in which PDGFR-ß expression was reduced below baseline levels, ie, in fewer than 25% to 30% of all cells, only minimal intimal thickening was observed. When PDGFR-ß expression exceeded baseline levels, intimal proliferation rose exponentially (Fig 4).

Although the first antisense sequence (AS1) almost completely reduced PDGFR-ß protein expression by day 14, it did not completely abolish intimal hyperplasia. This observation raises the possibility that although PDGFR-ßß stimulation may contribute up to 80% of the neointimal formation, the secretion of other growth factors or peptides might contribute to the residual fraction.^{32 33 34 35} Alternatively, the lack of complete inhibition of neointima may stem from the inability of the sustained antisense delivery system to fully suppress the immediate and early PDGF effect. The EVAc matrices allow the release of their embedded contents over the entire course of the experiment, not as a large bolus at the time of injury. On vascular injury, the almost-immediate platelet adhesion to subendothelial connective tissue induces the release of platelet PDGF-BB, which stimulates its PDGFR-ßß, and the interval of time between balloon denudation and oligonucleotide release on application may well have allowed sufficient growth factor–receptor interaction to activate the intracellular events that led to neointimal formation. Indeed, our in vitro study revealed first a complex pattern of PDGFR-ß protein expression in response to stimulation with PDGF-BB, with initial suppression of heightened baseline levels that returned within 48 hours, and second that pretreatment with AS1–PDGFR-ß oligonucleotide reduced receptor subunit expression at baseline by fourfold and on stimulation with PDGF-BB for the duration of the experiment (Fig 7). The administration of antisense PDGFR-ß oligomers days before the surgical procedure might reduce the baseline expression of PDGFR-ß sufficiently to prevent its interaction with PDGF-BB or the biological activity induction related to their interaction after the injury. Such studies could also allow one to determine the impact of these early interactions on residual intimal thickening.

The use of antisense technology is beset by questions of specificity.^{36 37 38} Recent reports have raised concern that the antiproliferative activity of antisense oligonucleotides to c-myb and c-myc, for example, arose from aptameric rather than a hybridization-dependent antisense mechanism.^{36 37} It was hypothesized that oligonucleotides with four sequential guanosines might bind to serum proteins, including growth factors such as bFGF, acidic fibroblast growth factor, PDGF, and vascular endothelial growth factor, reducing the interaction of these growth factors with their receptors and the intracellular signal transduction leading to gene protein expression (such as c-myc and c-myb) involved in cell-cycle progression.³⁸ Nonetheless, other studies have shown specific in vivo and/or in vitro effects of antisense oligonucleotides lacking multiple sequential guanosines on these and other genes involved in cell-cycle progression such as cdc2, cdk2, nonmuscle myosin, and PCNA.^{12 13 14 15 27} Neither antisense sequence used in the studies reported here possessed more than two contiguous guanosines. To more definitely address this issue, we examined the effects of the sequences on the -subunit. Because the antisense sequence can discriminate between oligonucleotide sequences that differ by one or two bases,^{16 39 40} we compared the effects of AS1 on the PDGFR- and PDGFR-ß. Quantitative analysis of protein expression on vSMCs in culture confirmed immunohistochemical identification of antigenicity in vivo. The antisense sequences directed against the PDGFR-ß inhibited only this targeted protein without affecting the PDGFR- protein expression (Figs 5 through 7). Scrambled oligonucleotide sequences also failed to reduce neointimal formation or PDGFR-ß protein expression in vitro or in vivo.

It is interesting to note that the antisense sequence closer to the 5' end of PDGFR-ß mRNA was more potent at inhibiting intimal thickening and PDGFR-ß protein expression than the AS2 sequence. This is in accordance with previous reports that showed that the biological effects of antisense oligomers are dictated in part by the location of the sense target sequence. Antisense oligonucleotides directed at or near the 5' translation initiation site were most effective at inhibiting gene expression, and in some cases, a shift of a few base pairs in the targeted sequence was sufficient to induce drastic variations in target gene inhibition.^{41 42 43 44} This discrepant effect between similar sequences remains enigmatic. A possible explanation could be that the secondary structure of the mRNA close to the initiation codon might offer a more favorable hybridization site for the antisense sequence. Downstream regions of the mRNA might fold and reduce the hybridizing access for the antisense sequences. Alternatively, antisense sequences complementary to or near the 5' mRNA region may be more potent at preventing mRNA translation.^{45 46 47} These and other issues require further study before antisense technology can reach its full potential.

Conclusions
In this study, we observed that the sustained perivascular application of antisense oligonucleotide sequences complementary to PDGFR-ß mRNA not only prevented overexpression of PDGFR-ß protein in healing medial and intimal vSMCs but did so in a manner commensurate with effects on intimal thickening. Almost complete abolition of PDGFR-ß protein expression was achieved with the antisense sequence closer to the 5' PDGFR-ß mRNA. The antisense PDGFR-ß effect was specific. The oligomers used did not bear four contiguous guanosines, eliminating concern for nonspecific, aptameric binding, and only the antisense sequences suppressed protein expression of only the target PDGFR-ß and not the PDGFR- protein.

Because PDGFR-ß expression is specific to mesenchymal cells such as vSMCs and fibroblasts, the regulation of this cell membrane receptor might provide an important advantage over the inhibition of cell-cycle proliferative proteins, which are expressed ubiquitously. Regulation of PDGFR-ß could contribute to the prevention of intimal thickening without affecting the proliferation of unrelated but critical cells. Further investigations are needed to determine whether and how the neointima will respond with release of PDGFR-ß protein expression inhibition after the removal or the degradation of the antisense oligomers. Finally, our results demonstrate again the value of antisense technology in helping to elucidate the mechanisms involved in vascular healing and as a possible approach to the prevention and progression of the accelerated arteriopathies that follow vascular intervention.

	Selected Abbreviations and Acronyms

 

bFGF = basic fibroblast growth factor EVAc = ethylene-vinyl acetate copolymer PCNA = proliferating cell nuclear antigen PDGF = platelet-derived growth factor PDGF-BB = PDGF ligand PDGFR-ß = PDGF receptor subunit PDGFR-ßß = PDGF receptor vSMC = vascular smooth muscle cell

	Acknowledgments

 
This study was supported in part by grants from the NIH, including GM/HL 49039 (Dr Edelman), and from the American Heart Association (CSA 9100420; Dr Simons), the Burroughs-Wellcome fund in Experimental Therapeutics (Dr Edelman), and the Whitaker Foundation for Biomedical Engineering (Dr Edelman). Dr Sirois was supported by a Canadian Heart and Stroke Foundation fellowship and is currently receiving a fellowship from the Medical Research Council of Canada.

Received May 7, 1996; revision received August 26, 1996; accepted September 19, 1996.

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