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(Circulation. 1998;97:1960-1969.)
© 1998 American Heart Association, Inc.

Basic Science Reports

PDGF-Receptor Tyrosine Kinase Blocker AG1295 Selectively Attenuates Smooth Muscle Cell Growth In Vitro and Reduces Neointimal Formation After Balloon Angioplasty in Swine

Shmuel Banai, MD; Yehuda Wolf, MD; Gershon Golomb, PhD; Andrew Pearle, BA; Johannes Waltenberger, MD; Ilia Fishbein, MD; Aviva Schneider, MS; Aviv Gazit, PhD; Louise Perez, BS; Rita Huber; Galila Lazarovichi; Laura Rabinovich, BPharm; Alexander Levitzki, PhD; ; S. David Gertz, MD, PhD

From the Heiden Department of Cardiology, Bikur Cholim Hospital (S.B.); the Departments of Vascular Surgery (Y.W.), Anatomy and Cell Biology (L.P., S.D.G.), and School of Pharmacy (G.G., I.F., L.R.), Hebrew University–Hadassah Medical School; the Departments of Biological Chemistry, Institute of Life Sciences (A.L., A.G.) and Organic Chemistry, Institute of Chemistry (A.G.), Hebrew University; the Joseph Lunenfeld Cardiac Surgery Research Center (A.S., G.L.), Jerusalem, Israel; and the Department of Internal Medicine II (Cardiology), Ulm University Medical Center, Germany (J.W., R.H.).

Correspondence to Shmuel Banai, MD, Department of Cardiology, Bikur Cholim Hospital, PO Box 492, Jerusalem 91004, Israel. E-mail banais{at}mail.netvision.net.il

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background—Signaling through protein tyrosine kinases (PTKs) is a major contributor to the transmission of mitogenic stimuli to the interior of the cell and nucleus. The present study was designed to determine the effect of the tyrphostin AG1295, a selective blocker of PDGF-receptor PTK, on the growth of porcine and human smooth muscle cells (SMCs) in culture, on the outgrowth kinetics of SMCs from porcine and human arterial explants, and on neointimal formation after balloon injury in pigs.

Methods and Results—SMCs for culture were obtained from porcine abdominal aortas, human internal mammary arteries, and endarterectomy tissue from a single human carotid artery. Addition of AG1295 to SMCs before PDGF stimulation completely inhibited PDGF-ß-receptor tyrosine phosphorylation without affecting the level of PDGF-ß-receptor. AG1295 resulted in a selective, reversible inhibition of SMC proliferation in culture (76%) with only mild (13.5%) inhibition of endothelial cell proliferation. The number of SMCs accumulating around explants of porcine carotid arteries and human endarterectomy specimens 12, 15, 19, 22, and 24 days after plating was reduced by 82% to 92% in AG1295-treated compared with nontreated specimens, and initiation of SMC outgrowth was markedly delayed. The numbers of cells accumulated 10 days after initiation of outgrowth were significantly lower in treated versus control explants. Local intravascular delivery of AG1295-impregnated polylactic acid–based nanoparticles (130±25 nm) to the site of balloon injury to porcine femoral arteries resulted in significant reductions in intima/media area ratio and luminal cross-sectional area narrowing by neointima compared with contralateral control arteries to which empty nanoparticles were applied (0.15±0.07 versus 0.09±0.03, P=.046 and 20±4% versus 10±4%, P=.0009, n=6 for both).

Conclusions—The tyrphostin AG1295, a selective blocker of PDGF-receptor kinase, exerts a marked inhibitory effect on the activation, migration, and proliferation of porcine and human SMCs in vitro and an 50% inhibitory effect on neointimal formation after balloon injury in porcine femoral arteries when delivered via biodegradable nanoparticles. Further studies appear to be warranted to evaluate the applicability of this novel approach to the interventional setting.

Key Words: muscle, smooth • tyrosine kinase • tyrphostin • platelet-derived factors • restenosis

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Proliferation and migration of activated SMCs, with release of abundant extracellular matrix by these cells, are fundamental to neointimal growth associated with accelerated arteriosclerosis, which continues to plague patients undergoing balloon angioplasty, coronary artery bypass surgery, and heart transplantation. A variety of experimental studies have been directed toward the attenuation of SMCs in vitro and in vivo. Nonetheless, relatively little progress has been made in the development of effective, selective, nontoxic inhibitors of SMC growth that might eventually be applied in the interventional setting. Recent progress in determining the mechanisms by which growth factors control cell proliferation has contributed to the development of treatment strategies that target specific signal transduction pathways to control proliferative disorders.^{1 2 3 4 5} The binding of specific growth factors with their selective cell surface receptor tyrosine kinases results in its autophosphorylation and activation, leading to downstream signal transduction through chains of intercommunicating proteins culminating in cell proliferation.^{6 7} Inhibitors of PTKs have been shown to suppress SMC chemotaxis and proliferation.^{8 9 10 11 12}

The tyrphostins are low-molecular-weight, synthetic compounds whose basic structure can be modified to block specific receptor PTKs or intracellular PTKs.^{3 13} Unlike larger receptor antibodies, the small size of the tyrphostins permits easier access to receptor sites within tissues such as those deep in the media. Recent studies have suggested that the profound selective PTK inhibition of such compounds results from competitive interaction with the ATP-binding domain as well as mixed competitive inhibition with substrate-binding subsites.^{14 15} Development of this class of compounds was based on the concept that it would lead to a more focused control of proliferative disorders, achieve more improved therapeutic indices, and reduce the numerous untoward side effects of the more generalized inhibitors of DNA or RNA synthesis or cytoskeleton-disrupting agents. We recently showed that controlled local delivery of the nonselective PTK blocker AG17 (RG50872) effectively inhibits neointimal formation in a rat carotid artery balloon injury model.¹⁶ The present study takes advantage of the selectivity of the described tyrphostin-type PTK inhibitor.

PDGF, expressed by platelets, SMCs, ECs, and macrophages, has been shown to play an important role in the pathogenesis of injury-induced neointimal formation in the arterial wall, acting as both a mitogen and chemoattractant for SMCs as well as being involved in the transformation of SMCs from their contractile to the proliferative phenotype.^{17 18} In vivo studies have demonstrated that the expression of PDGF ligand and its receptor is elevated after arterial injury.¹⁹ Infusion of PDGF into injured rat carotid arteries and transfection of a plasmid coding for PDGF into pig arteries have also been shown to increase neointimal formation.^{20 21} PDGF receptor levels in SMCs from human atherosclerotic plaques have also been reported to be elevated compared with receptor levels in normal medial SMCs.²² Recently, Sirois et al²³ showed marked upregulation of PDGF receptors after injury to the vessel wall. They have demonstrated that the degree of neointimal formation substantially depends on both PDGF-ß-receptor overexpression and its activation by PDGF-BB. They demonstrated further that controlled local delivery of antisense oligonucleotides to PDGF-ß receptor reduces neointimal formation in the rat carotid injury model. Finally, PTK blockers of the tyrphostin family have been shown to block PDGF-receptor signal transduction, including the phosphorylation and activation of phospholipase C-, believed to be involved in SMC migration.^{11 12 24 25} We therefore hypothesized that selective blockade of PDGF-ß-receptor activation should also result in marked inhibition of SMC activation, migration, and proliferation.

We show here that the tyrphostin AG1295, a selective blocker of PDGF-receptor PTK, inhibits PDGF-BB–induced PDGF-ß-receptor phosphorylation without affecting receptor protein levels, selectively inhibits porcine and human SMC proliferation in culture with only a minimal effect on ECs, attenuates the outgrowth of SMCs from porcine and human arterial explant tissue in vitro, and inhibits neointimal formation after balloon injury in pigs by 50% after local, controlled, intravascular delivery of biodegradable nanoparticles.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Effect of AG1295 on PDGF-Induced Receptor Autophosphorylation in Intact Cells
Subconfluent porcine arterial SMCs cultivated in DMEM supplemented with 15% FCS were synchronized for 20 hours in medium containing 2% FCS. After preincubation with AG1295 (10 µmol/L) for 60 minutes and with Na₃VO₄ (100 µmol/L) for 5 minutes, the cells were stimulated with PDGF-BB (100 ng/mL) for 10 minutes at 37°C. The cells were solubilized in NP-40 (1%) lysis buffer. The analysis of PDGF-ß-receptor phosphorylation was performed as described previously.²⁶ Briefly, cell lysates were used for immunoprecipitation with the PDGF-ß-receptor–specific antiserum R3, and the samples were subjected to SDS-PAGE (7.5%) for receptor analysis and blotted onto a nitrocellulose membrane (Hybond C extra, Amersham). Phosphorylated proteins were detected by immunoblotting with the horseradish peroxidase–conjugated phosphotyrosine antibody RC20H (Transduction Laboratories), followed by application of a chemoluminescence-based detection system (ECL, Amersham) and autoradiography. Detection of receptor proteins was performed in a similar way by immunoblotting with the specific R3 antiserum followed by several washing steps and the application of a horseradish peroxidase–conjugated donkey anti-rabbit antibody (Amersham) and visualization with chemoluminescence and autoradiography as described above.

Cell Culture Techniques
SMCs were obtained under aseptic conditions from 6 pig abdominal aortas, 6 human internal mammary arteries, and endarterectomy tissue from a single human carotid artery by the explant technique.^{27 28 29} Specimens from the operating room were transferred on ice to the tissue culture room, where each artery was cut open and the endothelial surface mechanically scraped. The vessels were then cut into 2-mm² fragments, which were placed in culture dishes with DMEM supplemented with 15% (vol/vol) FCS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.2 mol/L L-glutamine. The medial tissue fragments were then placed in an incubator at 37°C in 9% CO₂ until SMC outgrowth was detected. Uniform populations of SMCs that displayed the characteristic "hill-and-valley" growth pattern were subcultured with 0.25% trypsin. For experiments testing the effect of AG1295 on growth inhibition and reversibility, SMCs from passages 1 to 3 were replated on 15-mm wells pretreated with 3 µg/cm² fibronectin^{30 31} (Biological Industries) at 15 000 cells/well.

ECs were isolated from porcine carotid arteries.^{32 33} Under aseptic conditions, both common carotid arteries were isolated, and the distal end of each artery was cannulated through an arteriotomy and ligated. The arteries were then perfused with PBS, and the proximal end was ligated, isolating a 5- to 7-cm-long blood-free portion of the artery. The isolated portion of each artery was filled with PBS containing calcium and magnesium with 0.1% collagenase. The segments were excised and incubated for 10 minutes at 37°C in sterile bottles containing PBS. The arterial effluent was then flushed out with medium (M199 supplemented with 15% FCS, penicillin 100 U/mL, streptomycin 100 µg/mL, 0.2 mol/L L-glutamine, and 25 µg/mL ECGS [Biomedical Technologies, Inc]) and collected in 50-mL centrifugation tubes containing 5 mL of medium. The cell suspension was centrifuged (200g, 5 minutes) and the pellet resuspended in culture medium. Cells were seeded on fibronectin-coated dishes at a seeding density of 15 000 cells/well and incubated at 37°C in 9% CO₂. The ECGS (25 µg/mL) was added every other day until confluence. At confluence, the cells were removed with trypsin-EDTA solution (0.25% trypsin plus EDTA 1:2000 in Puck's saline), resuspended in culture medium, counted, and replated at 15 000 cells/well in fibronectin-coated four-well dishes (15 mm) for the growth inhibition experiments.

Inhibition of Cell Proliferation and Reversibility
Monolayer cell growth inhibition and reversibility experiments were repeated three or four times, with each experiment having been performed in triplicate. Approximately 15 000 cells (SMCs or ECs) in 1 mL of culture medium supplemented with 15% FCS were seeded on day 0 in 15-mm wells precoated with fibronectin. Cultures were treated with AG1295 (10 µmol/L) dissolved in 0.1% DMSO on days 1 and 3. On day 6, cultures were washed and the cells allowed to recover. Cells were counted on days 3 and 5 for inhibition and on days 7, 10, and 15 for reversibility. The medium supplemented with serum (M199 with ECGS for ECs and DMEM for SMCs) was changed every other day. The effect of AG1295 on cell proliferation was compared with three control groups: (1) DMSO (0.1%) without AG1295; (2) medium with serum only; and (3) AG17 (10 µmol/L), a potent, nonselective PTK blocker.^{5 34 35 36}

Arterial Explant Techniques
Explant tissue was obtained from six porcine common carotid arteries and human atheroma retrieved from a single patient undergoing carotid endarterectomy. The specimens were placed in PBS with penicillin (100 U/mL)/streptomycin (100 µg/mL). After a washing in additional PBS, the endothelium was removed by gentle scraping, and the adventitia was peeled off with fine forceps. The medial specimens were cut into 1-mm² fragments with a sharp scalpel blade. To measure cellular accumulation around explants, four fragments were placed in four-well dishes pretreated with fibronectin. For outgrowth initiation and outgrowth index studies, 96-well plastic culture dishes were pretreated with fibronectin, and an individual fragment was placed in each well. Explants were left undisturbed for 45 minutes without growth medium at room temperature to allow for explant attachment. Fragments in the four-well plates were then immersed in 1 mL of culture medium supplemented with 7.5% human serum and 7.5% FCS (human endarterectomy specimens) or 15% FCS (porcine arterial specimens). Fragments in 96-well plates were immersed in 150 µL of the appropriate culture medium. The plates were placed in a humidified incubator (5% CO₂) at 37°C, and the medium was changed every 2 days. AG1295 (50 µmol/L dissolved in 0.5% DMSO) was added to the culture medium of treated explants every 2 days throughout the experiment. An equal concentration of DMSO was added to the medium of control explants. Samples of arterial explants for histological evaluation were taken before and immediately after removal of the endothelium. These samples were fixed in 4% buffered formaldehyde, dehydrated in ethanol and xylene, and embedded in paraffin. Sections (5 µm) were stained with hematoxylin-eosin and by the Movat technique.³⁷

The overall accumulation of SMCs around each explant was measured at 12, 15, 19, 22, and 24 days after plating. Experiments were performed in triplicate so that for each of the five time periods tested there were three wells containing four explants, each yielding 12 treated and 12 control explants, for a total of 120 explants. Each experiment was repeated twice. At each of the five time periods after plating, the explants were removed from the wells, and the cells that had grown out from the tissue and accumulated on the plate were enzymatically dispersed (0.25% trypsin, 1 mmol/L EDTA) and counted in a Coulter Counter. The accumulation of SMCs around the explants was expressed as the total number of cells per well.

The time course of the initiation of outgrowth was determined with 95 additional control and 96 treated porcine carotid explants and 23 control and 23 treated explants of human atheroma specimens (all from the same individual). The specimens were scored every other day by two independent observers to determine the number of explants yielding outgrowth of SMCs. Explants showing two or more SMCs at the edges of the tissue were counted as positive for outgrowth initiation. The time course of outgrowth (percentage of explants yielding outgrowth per days in culture) was plotted. The porcine carotid and human endarterectomy explants were followed until the proportion of explants with outgrowth was constant (21 and 34 days, respectively).

To establish the rate of proliferation of SMCs after their activation in control versus treated explants, an index of outgrowth was determined by counting the number of SMCs that had grown around each explant 10 days after outgrowth was first observed. This index excludes the lag time before outgrowth initiation and thereby permits the estimation of the overall rate of SMC proliferation in cells already activated. The cells that had proliferated around each explant at this time were enzymatically dispersed, pooled, and counted.

Immunocytochemistry
Identification of SMCs in the outgrowth phase and in the first subculture was confirmed by -actin staining of primary outgrowth and passaged cells. The cells were fixed in 4% paraformaldehyde and immunostained with mouse monoclonal antibodies directed against -smooth muscle actin (Mouse Monoclonal, Sigma Chemical Co, product No. 6582, clone 1A4). The secondary antibody, peroxidase-conjugated Affinipure goat anti-mouse IgG (heavy and light chains) (Jackson Immuno Research Laboratories), was visualized by incubation with an AEC chromagen (peroxidase chromagen C3-amino,9-ethyl carbazole; Biomeda Corp).

Neointimal Formation After Balloon Injury In Vivo
Eight juvenile domestic swine (15 to 20 kg) were sedated by intramuscular injection of 1% propionylpromazin (0.1 mL/kg). Anesthesia was induced with ketamine hydrochloride (20 mg/kg IM) and droperidol (0.2 mg/kg IM) followed by 6% sodium pentobarbital (0.25 mL/kg IV). After endotracheal intubation, the pigs were ventilated with a mixture of oxygen and room air. After surgical exposure of the CFAs and the proximal portions of the superficial femoral arteries bilaterally, all side branches of the CFA were ligated. After administration of heparin (5000 U IV bolus), a high-torque floppy angioplasty guidewire (0.014 in) was inserted into the CFA through an arteriotomy in the superficial femoral artery, followed by over-the-wire passage of the balloon angioplasty catheter (3.0 to 3.5 mm in diameter, noncompliant, 20 mm long, balloon-to-artery ratio, 1.5:1). The balloon was then inflated in the CFA and withdrawn under tension (7 to 8 atm). After five passes, the balloon was kept inflated in the CFA for 2 minutes. All balloon injuries were performed by the same investigator. After deflation, the balloon was removed, an infusion catheter was inserted over the wire into the CFA, and the wire was removed. The injured segment of the CFA was isolated by proximal and distal occlusion with Yasargil atraumatic arterial clips. Polylactic acid–based nanoparticles (130±25 nm), prepared by emulsification evaporation, with or without AG1295 (90 to 110 µg/mL) were delivered into the isolated injured segment (0.3- to 0.4-mL volume). The solution was retained within the isolated segment for 30 minutes (AG1295-impregnated ipsilaterally or bare nanoparticles contralaterally). After withdrawal of the solution, the clamps and infusion catheter were removed, flow was restored, and the superficial femoral artery was tied. The presence of nanoparticles within the arterial wall after this procedure was confirmed in two additional arteries by high-performance liquid chromatography 24 hours after restoration of blood flow. After closure of the skin, the animals were allowed to recover and were returned to their pens. One animal was found dead in its pen after the surgery, and a second was excluded because of surgical mishap on the sham-operated control side. Four weeks later, under general anesthesia and mechanical ventilation, both femoral arteries were exposed at the site of balloon injury. The abdominal aorta and inferior vena cava were isolated, ligated, and cannulated at the level of the renal vessels. The animals were euthanized with sodium pentobarbital (60 mg/kg) followed by a rapid bolus of KCl (40 mEq/L IV). The arteries were flushed via the aortic cannula with normal saline (1000 mL with 3 mL heparin [5000 U/mL], 37°C, 90 mm Hg) and pressure-perfused with 4% buffered formaldehyde (1000 mL, 37°C, 90 mm Hg). The perfusion effluent was drained via the inferior vena cava cannula. Segments of the right and left CFAs were excised, cut into 1- to 2-mm segments, and embedded in paraffin. Cross sections 4 µm thick were stained by the Movat pentachrome technique. Computerized morphometric analysis was performed on all sections with a CUE-2 image analyzer (Galai Productions, Ltd) in association with an Olympus BH-2 microprojection system. The areas measured were total area bounded by the external elastic lamina (EEL area), area bounded by the IEL (IEL area), and area occupied by the lumen (LU area). Derived measurements of neointimal formation included the I/M ratio (IEL area-LU area÷EEL area-IEL area) and the %CSAN-N ([IEL area-LU area]x100÷IEL area).

Statistical Analysis
Results are expressed as mean±SD. For the in vitro studies, the effect of the various doses of tyrphostins versus control, at any time period, was assessed by one-way ANOVA with Fisher's protected least significant difference as the post hoc test. Comparisons between tyrphostin treatment and control for individual morphological parameters at one specific time point were assessed by unpaired, two-tailed t test. Comparisons between tyrphostin treatment and control for individual morphological parameters over multiple time points were assessed by two-factor ANOVA. Histomorphometric comparisons were made on the section most narrowed by neointima from each artery. The differences in I/M ratio and %CSAN-N after balloon injury in vivo between AG1295-treated and contralateral sham control arteries were determined by the paired two-tailed t test. The Statview II statistical package (Brain Power, Inc) was used for these calculations.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Inhibition of PDGF-Induced Receptor Autophosphorylation
Stimulation of porcine arterial SMCs with PDGF-BB (100 ng/mL) resulted in strong phosphorylation of the PDGF-ß-receptor on tyrosine residues. Addition of AG1295 to the cells before PDGF stimulation completely inhibited PDGF-ß-receptor tyrosine phosphorylation (Fig 1A). AG1295 did not affect the level of PDGF-ß-receptor protein present in the cells (Fig 1B).

View larger version (28K): [in this window] [in a new window]   Figure 1. Immunoblot analysis. Effect of AG1295 on PDGF-BB–induced tyrosine phosphorylation of PDGF-ß-receptor in intact porcine arterial SMCs (A) as well as levels of PDGF-ß-receptor expression (B). Pretreatment and stimulation of porcine arterial SMCs was followed by immunoprecipitation of PDGF-ß-receptor (R3 antiserum), SDS-PAGE (7.5%), and immunoblot analysis (RC20H in A; R3 in B). Detection of hybridizing antibodies was obtained with a chemoluminescence-based detection system (ECL, Amersham).

Cell Culture Studies of Enzymatically Dispersed Cells
Porcine Aortic SMCs
Treatment with AG1295 resulted in a 46% mean reduction in SMC count by day 3 compared with DMSO-treated control cells and a 76±2% (mean±SD) reduction over control by day 5. The nonselective PTK blocker AG17 inhibited SMC growth by 79% and 91±2% at days 3 and 5, respectively. Whereas the effect of AG17 was not reversible and cells did not resume proliferation after treatment was withdrawn, the inhibitory effect of AG1295 was completely reversible (Fig 2).

View larger version (39K): [in this window] [in a new window]   Figure 2. Dose response and reversibility of inhibitory effect of AG1295 on porcine SMC proliferation. For this experiment, cells were treated with AG1295 on days 1 and 3. On day 7, cultures were washed and cells allowed to recover. Cells were counted on day 7 for inhibitory effect and on day 15 for recovery. AG1295 1, 5, and 10 µmol/L caused 23%, 51%, and 64% inhibition of proliferation, respectively. In all doses tested, effect was not toxic and reversible. *P<.05, **P<.001 vs control by one-way ANOVA.

Porcine ECs
The inhibitory effect of AG1295 on EC proliferation was minimal, resulting in only a 10% mean reduction of cell growth by day 3 and a 13.5±3% reduction by day 5 compared with control ECs (Fig 3). This mild inhibitory effect was completely reversible. The nonselective AG17 resulted in a 55% and 91±12% mean reduction of EC growth by days 3 and 5, respectively (Fig 3), and this effect was not reversible after treatment was discontinued.

View larger version (42K): [in this window] [in a new window]   Figure 3. Degree of inhibition of cell proliferation by AG17 and AG1295. Note that AG17, the nonselective PTK blocker, inhibited both SMC and EC proliferation (mean, 91% inhibition for both ECs and SMCs), but AG1295 selectively inhibited SMC proliferation (mean, 76% inhibition) with only a mild inhibitory effect on EC proliferation (mean, 13.5%). Cells were treated with tyrphostins on days 1 and 3 and counted on day 5.

Human Internal Mammary Artery SMCs
Treatment with AG1295 resulted in a 50% mean reduction in SMC proliferation by day 3 and a 72% mean reduction by day 5 compared with untreated or DMSO-treated cells. This effect was completely reversible (Fig 4A).

View larger version (13K): [in this window] [in a new window]   Figure 4. Inhibitory effect of AG1295 on human internal mammary artery (A) and human atheroma–derived SMC (B) proliferation and reversibility of the effect. Mean inhibitory effect during treatment days 3 and 5 was 64% and 74%, respectively, for human atheroma–derived cells and 50% and 72% for human internal mammary artery–derived cells. P<.0001 and P<.003, respectively, by two-way ANOVA.

Human Atheroma-Derived SMCs
AG1295 inhibited human atheroma–derived SMC growth by 64% and 74% by day 3 and 5, respectively, compared with untreated or DMSO-treated cells. This effect was completely reversible (Fig 4B).

Arterial Explant Studies
Outgrowth from porcine carotid artery and human carotid endarterectomy explants began at the margins of the specimens 4 and 8 days after plating, respectively. The first cells that migrated out of the margins of the explanted tissue were morphologically and immunohistochemically indistinguishable between treated and control wells. These cells were elongated and spindle-shaped, and only a small percentage (1% to 5%) stained positive for filamentous -actin within the first 5 days after outgrowth initiation in both treated and control explants. These cells assumed the well-known hill-and-valley configuration often attributed to the proliferative phenotype of SMCs in culture. However, after reaching confluence (10 days after outgrowth initiation), the cells appeared to redifferentiate, and the percentage of SMCs that stained positive for filamentous -actin reached 50% to 70%. In general, the more distant cells from the explant exhibited more intense -actin staining in both treated and control specimens. A much greater percentage of SMCs from AG1295-treated explants (versus control) assumed the larger, polygonal, and well-spread profile with numerous -actin–positive stress fibers often attributed to the contractile phenotype. The morphological features of SMCs from AG1295-treated explants were similar to those of SMCs seen in monolayers of passaged cells (Fig 5). The explanted tissue in the control wells appeared to shrink as the cells migrated out of the explant, but shrinkage was not apparent in the treated tissue.

View larger version (77K): [in this window] [in a new window]   Figure 5. SMC accumulation at margins of porcine carotid explants 5 days after outgrowth initiation. Note that SMCs growing out from control explants (top) appear more numerous and are more spindle-shaped, with fusiform nuclei and less prominent -actin staining. These cells are rapidly proliferating. Fewer cells are seen around explants treated with tyrphostin AG1295 (middle). These SMCs appear larger, with a more spread, polygonal morphology and intense -actin–positive fibers (x600). Bottom, Pig SMCs treated with AG1295 (x1200).

Histological evaluation of the hematoxylin-eosin–stained and Movat-stained sections of the explants that underwent luminal scraping showed complete desquamation of the endothelium with the virtual absence of the IEL. The IEL was intact in explants in which the endothelium had not been scraped off.

Outgrowth Kinetics From Explant Tissue
Initiation of Outgrowth
Treating porcine carotid explants with AG1295 resulted in a marked prolongation of the time between the plating of the arterial tissue and the appearance of cells around the explants. In control explants, 36% of the 95 explants showed SMC growth initiation 7 days after plating. In contrast, in the tyrphostin-treated specimens, only 12% of the 96 explants had cells at their margins at this time. Outgrowth was seen in 50% of control specimens by day 8, whereas outgrowth in 50% of the treated specimens was observed only on day 12. The outgrowth reached a plateau on day 12 in the control tissue (96% to 100%) but not until day 19 in the treated samples (91% to 95%) (Fig 6A).

View larger version (16K): [in this window] [in a new window]   Figure 6. Delay in outgrowth initiation of SMCs caused by AG1295 from porcine arterial explants (A) (P<.05) and from human atheroma explants (B) (P<.0001) (two-way ANOVA).

The human carotid endarterectomy specimens showed similar outgrowth kinetics and response to tyrphostin treatment. Seventeen percent of control explants showed SMC outgrowth initiation at day 10, but at this time, none of the treated specimens had outgrowth (Fig 6B). Likewise, by day 14, 50% of control explants had outgrowth, but in tyrphostin-treated specimens, 50% outgrowth was seen only after day 23. As with the porcine specimens, a delay in the time to reach the plateau was also found in the human tissue. In the untreated human control specimens, SMC outgrowth became constant at 91% by day 22, but in the treated specimens, this plateau was reached only at day 30, and the percentage of specimens showing outgrowth at plateau was only 61%.

Outgrowth Index
The mean number of SMCs that accumulated around porcine carotid explants 10 days after outgrowth initiation was first observed was 70% lower in those treated with AG1295 than in control samples (8489±1764 versus 28 626±2977, P<.00001) (Fig 7A). Likewise, the mean SMC accumulation 10 days after onset of outgrowth from human atheroma specimens was significantly lower in explants treated with AG1295 than in control samples (6937±704 versus 18 945±6943, P=.0001) (Fig 7B). This represents a 63% inhibition of SMC accumulation around the treated explants.

View larger version (30K): [in this window] [in a new window]   Figure 7. Outgrowth index of SMCs from porcine carotid artery explants (A) and human endarterectomy specimens (B). Note marked inhibitory effect of AG1295 on SMC outgrowth and accumulation around explants 10 days after initiation of outgrowth in each explant. * P<.001 by unpaired, two-tailed t test.

Overall Accumulation of SMCs Around Porcine Carotid Explants
The total number of cells accumulating around control explants increased with time. However, in explants treated with AG1295, SMC growth was less by 82% to 92% at all times (Figs 8 and 9).

View larger version (25K): [in this window] [in a new window]   Figure 8. Inhibitory effect of AG1295 on SMC accumulation around porcine explants measured 12, 15, 19, 22, and 24 days after plating (total number of cells per well). Inhibition of cell accumulation (AG1295-treated vs control) was between 82% and 92% at all time points (P<.0001 by two-way ANOVA).

View larger version (139K): [in this window] [in a new window]   Figure 9. Porcine arterial SMC accumulation around carotid arterial explants (24 days after plating of explant). Note significantly fewer cells growing out of AG1295-treated explant (top) vs control explant (bottom) (see Fig 8). Morphological differences between treated and untreated cells are seen in higher magnification in Fig 5. E indicates site of explant that was removed before photography. -Actin stain (x300).

Neointimal Formation After Balloon Injury In Vivo
Local intravascular delivery of AG1295-impregnated polylactic acid–based nanoparticles to the site of controlled balloon injury to porcine femoral arteries resulted in a significant reduction in I/M ratio compared with contralateral control arteries to which empty nanoparticles were applied (0.15±0.07 versus 0.09±0.03, P=.046, n=6 for both) (Fig 10). The utility of the I/M ratio for assessing neointimal narrowing across arterial samples presupposes consistency in medial area and overall vessel wall size. Whereas the medial areas were very consistent (176±14x10⁴ µm²), the overall vessel wall size showed a slightly greater variability (313±60x10⁴ µm²). For this reason, %CSAN-N was also used (see "Methods"), which measures the degree to which the IEL area is reduced by neointima and normalizes, to a great degree, the effect of changes in vessel wall size. The mean %CSAN-N at sites of balloon injury of arteries to which AG1295-impregnated nanoparticles were delivered was significantly less than contralateral control arteries (10±4% versus 20±4%, P=.0009). Inflammatory cell infiltrate within the intima and media at sites of nanoparticle delivery was relatively light or nondetectable, and no difference was detected between AG1295-impregnated and empty nanoparticles. Inflammatory cell infiltrate within the adventitia did not appear to depart from that seen in angioplastied porcine arteries not subjected to this intravascular delivery.

View larger version (142K): [in this window] [in a new window]   Figure 10. Porcine femoral arteries 28 days after balloon injury showing marked reduction in neointimal area after intravascular delivery of AG1295-impregnated nanoparticles (top) compared with sham-operated control artery (bottom) that received empty nanoparticles. Movat stain (x220).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
These experiments demonstrate that tyrphostin-mediated inhibition of the PDGF-ß-receptor autophosphorylation results in the selective inhibition of SMC activation, proliferation, and migration in vitro, with a minimal effect on ECs and a significant reduction of neointimal formation in vivo in a pig balloon injury model. The tyrphostin AG1295 completely inhibited the PDGF-BB–induced phosphorylation of the PDGF-ß-receptor tyrosine residues of porcine arterial SMCs without affecting the level of PDGF-ß-receptor protein present in these cells, providing additional support of effective inhibitory activity without significant toxicity. Although it has been shown that this tyrphostin is a highly selective blocker of the PDGF-receptor PTK,¹⁵ given the large number of known protein kinases, the possibility that AG1295 may display some activity against other kinases cannot be excluded.

Because in vitro findings from passaged SMCs may be too far removed from the in vivo situation to reflect the biological properties of SMCs in the vessel wall, we used the arterial explant model as a "bridge" between the in vitro cell culture experiments and the in vivo porcine balloon injury experiments. The explant model permits the ex vivo study of SMC transformation, migration, and proliferation in a system that preserves many but certainly not all aspects of the arterial tissue relationships and microenvironment, including the variety of local paracrine and autocrine systems.³⁸ Outgrowth initiation is the first end point in this model. The time for the first cell to appear at the margins of the explant is a marker for tissue activation, because it represents the first appearance of a transformed population of cells able to migrate out of the tissue and proliferate. The second end point is the accumulation of cells around each explant 10 days after the appearance of the first cell. This end point controls for variations in the lag time for activation in each explant. With this model, AG1295 markedly reduced the total number of cells accumulating around the explanted porcine and human arterial specimens, prolonged the time to initiation of outgrowth, and delayed the time to reach the growth plateau. The outgrowth index, calculated from the total number of cells present 10 days after the initiation of outgrowth for each specimen, a parameter that spans the logarithmic phase of SMC proliferation, was also markedly reduced.

The determination of the overall number of SMCs accumulating around the explants at various time points after plating does not discern the relative role of activation, migration, and/or proliferation but is a straightforward method of assessing the combined effects of these important cellular events. The effects of AG1295 on SMC activation and proliferation were more specifically observed from the significant delay in the initiation of SMC outgrowth from the explants, the prolongation of time to attainment of the growth plateau, and the marked effect on the outgrowth index. The effect of this agent was also apparent from the morphological and immunohistochemical observations showing that AG1295 seemed to maintain the SMCs in a contractile and predominantly nonproliferating phenotype even though the tissue was subjected to explantation and exposed to serum mitogens.

The marked inhibitory effect of AG1295 on SMCs in vitro was confirmed in vivo by intravascular delivery of tyrphostin-impregnated biodegradable nanoparticles to the site of balloon angioplasty in porcine femoral arteries. The vast majority of cells in the media of healthy, uninjured adult arteries are SMCs. Injury to the vessel wall, with loss or damage to the endothelium, causes a subpopulation of the quiescent, differentiated SMCs to lose their contractile myofilamentary apparatus and transform into synthetic cells with large amounts of rough endoplasmic reticulum, ribosomes, and mitochondria. This transformation, directed at least in part by PDGF, is associated with SMC migration and proliferation followed by elaboration of abundant extracellular matrix. The signal transduction induced by PDGF-BB, considered by many to be the strongest known mitogen and chemoattractant for arterial SMCs, stimulates directed migration and proliferation of arterial SMCs into the neointima after arterial injury.³⁹ It has been suggested that if the endothelium regenerates rapidly after injury, the synthetic SMCs return to a contractile, nondividing phenotype, and neointimal formation is reduced.⁴⁰ If, however, the injury is severe or sustained, the cells may remain in their synthetic-proliferative phenotype and retain their heightened responsiveness to mitogens. PDGF receptors are thought to be expressed primarily in SMCs, whereas vascular endothelial growth factor receptor expression is considered to be restricted largely to the endothelium. The novel approach to the inhibition of neointimal formation by AG1295 presented in the present study takes advantage of the marked selective, nontoxic inhibition of SMC PDGF-ß-receptor kinase, with virtually no effect on the kinase activity of the vascular endothelial growth factor receptor.¹² By a different methodological approach, Chang et al⁴¹ recently reported inhibition of SMC proliferation in vitro and in vivo in a rat carotid artery model of balloon injury by adenovirus-mediated overexpression of the cyclin-dependent kinase inhibitor p21, which blocks the initiation of the S phase of the cell cycle and inhibits proliferating cell nuclear antigen, but further studies are necessary to determine the selectivity of this regimen across cell types and species. Recent experiments using antisense oligonucleotides and neutralizing antibodies further support the concept of PDGF-ß-receptor blockade as a treatment strategy to inhibit neointimal formation and premature arterial stenosis.⁴² The small molecular size of the tyrphostins, such as AG1295, used in the present study has the additional advantage of permitting easier access to SMC-PDGF receptors within the media and adventitia, and the use of small, biodegradable nanoparticles as the delivery vehicle provides for prolonged intramural exposure to the site of injury.

In conclusion, these studies demonstrate a profound effect of the tyrphostin AG1295 on the outgrowth kinetics of SMCs in culture and explant tissue and a marked inhibitory effect on neointimal formation after balloon injury in vivo. On the basis of these results, additional studies appear to be warranted to determine the long-term effects of intravascular delivery of tyrphostin-impregnated biodegradable nanoparticles on the arterial wall to evaluate the applicability of this novel approach to the interventional setting.

	Selected Abbreviations and Acronyms

 

CFA = common femoral artery %CSAN-N = percent luminal cross-sectional area narrowing by neointima EC = endothelial cell ECGS = endothelial cell growth substitute IEL = internal elastic lamina I/M = intima to media area PDGF = platelet-derived growth factor PTK = protein tyrosine kinase SMC = smooth muscle cell

	Acknowledgments

 
This work was supported by the Joint Fund of the Israel Ministry of Science and Arts and The German Ministry of Science, Technology, and Education (BMBF) DISMED87/1338GR. Mr Pearle was supported by the Stanford Medical Student Scholars Program, Stanford School of Medicine. The work of Prof Levitzki was supported in part by a grant from the Sugen Corp, Redwood City, Calif. The authors wish to thank Prof Joseph Borman and Prof Gideon Merin for making available the facilities of the cardiac surgery research laboratory. Dr Gertz holds the Lillian and Rebecca Chutick Chair of Cardiac Studies, The Hebrew University, Jerusalem.

Received July 23, 1997; revision received November 18, 1997; accepted December 12, 1997.

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