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Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:667-676

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:667.)
© 2000 American Heart Association, Inc.

Vascular Biology

Local Delivery of Platelet-Derived Growth Factor Receptor–Specific Tyrphostin Inhibits Neointimal Formation in Rats

Ilia Fishbein1; Johannes Waltenberger1; Shmuel Banai; Laura Rabinovich; Michael Chorny; Alexander Levitzki; Aviv Gazit; Rita Huber; Ulrike Mayr; S. David Gertz; Gershon Golomb

From the Department of Pharmaceutics (I.F., L.R., M.C., G.G.), School of Pharmacy, Faculty of Medicine, the Department of Biological Chemistry (A.L., A.G.), Institute of Life Sciences, and the Department of Anatomy and Cell Biology (S.D.G.), The Hebrew University of Jerusalem, Jerusalem, Israel; the Department of Internal Medicine II (Cardiology) (J.W., R.H., U.M.), Ulm University Medical Center, Ulm, Germany; and the Department of Cardiology (S.B.), Bikur Cholim Hospital, Jerusalem, Israel.

Correspondence to Dr Gershon Golomb, Department of Pharmaceutics, School of Pharmacy, The Hebrew University of Jerusalem, POB 12065, Jerusalem 91120, Israel. E-mail golomb{at}cc.huji.ac.il


*    Abstract
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Abstract—Signal transduction through the platelet-derived growth factor (PDGF)/PDGF receptor (PDGFR) system is involved in the process of postangioplasty restenosis. Tyrphostins are low molecular weight inhibitors of protein tyrosine kinases. We assessed the antiproliferative effects of PDGFRß-specific tyrphostin AG-1295 in vitro and in vivo. AG-1295 significantly inhibited rat smooth muscle cell growth stimulated by PDGF-BB or FCS. This antiproliferative effect was paralleled by reversible reduction of the total phosphotyrosine level and the degree of PDGFRß phosphorylation by the drug in vitro. Local sustained delivery of the drug from perivascularly implanted polymeric matrices resulted in focal AG-1295 levels of 711 and 29.1 ng/mg of dry arterial tissue 1 and 14 days after implantation in rats. AG-1295 delivered from polymeric matrices resulted in a 35% reduction of neointimal formation on day 14 after balloon injury in the rat carotid model. Tyrosine phosphorylation of certain transduction proteins in arterial tissue extracts was significantly upregulated by balloon injury on day 3 but was essentially returned to or below basal levels 14 days after injury. Tyrphostin treatment decreased tyrosine phosphorylation at both time points below the basal levels. Moreover, the enhancement of PDGFRß expression 3 and 14 days after arterial injury was strongly inhibited by AG-1295 treatment. It can be concluded that AG-1295 reduces neointimal formation by inhibiting PDGFß-triggered tyrosine phosphorylation.


Key Words: restenosis • protein tyrosine kinase • controlled release • platelet-derived growth factor • tyrphostins


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Arterial injury inflicted by percutaneous transluminal coronary angioplasty triggers the development of neointimal formation that obviates an initial gain obtained during dilation of stenotic vessels or stent placement. When it occurs in association with unfavorable remodeling, this process leads to clinically significant sequelae.

Arterial neointima contains smooth muscle cells (SMCs) with altered growth characteristics and dense extracellular matrix elaborated by activated SMCs.1 Normal arterial intima is essentially devoid of SMCs, and repopulation of intima by {alpha}-actin–positive cells is an important step in the pathophysiology of restenosis.2 Migration of medial SMCs3 and adventitial myofibroblasts4 and their consequent intimal proliferation are governed by the combined effects of several growth factors released from aggregated platelets, injured endothelium, and activated SMCs.1 5 Of all growth factors and cytokines involved, platelet-derived growth factor (PDGF) is the one most intimately implicated in the process of intimal SMC accumulation.6 In contrast to its potent stimulation of proliferative activity on SMCs in vitro,7 PDGF apparently plays no major role in the first wave of SMC proliferation in the media after arterial injury.8 9 10 Thus, the current paradigm of PDGF involvement in neointimal formation is based on migration enhancement of the medial SMCs to the neointima.1 11

Signaling cascades, initiated by the binding of PDGF to its cognate receptors, are meditated by the intrinsic protein tyrosine kinase (PTK) activity of receptors and several cytoplasmic checkpoint proteins.12 13 14 Migration and proliferation of vascular SMCs critically depend on intact signal transduction through PTK.15 Although it has recently been shown16 17 that pathways for PDGF-driven SMC proliferation and migration bifurcate at an early point, they still share a common proximal section. Thus, inhibition of the autocrine/paracrine loop of PDGF could be achieved through the inhibition of PDGF receptor (PDGFR) tyrosine kinase activity. It was shown that a series of low molecular weight PTK inhibitors, termed tyrphostins (for review, see References 18 and 1918 19 ), inhibit PDGF-dependent DNA synthesis and cell growth in cultured rabbit vascular SMCs20 and proliferation and migration in cultured rat aortic SMCs.21 22 23 This inhibition correlates with the potency of tyrphostins to inhibit the PDGF-dependent tyrosine autophosphorylation of intracellular substrates and the activation of phospholipase C{gamma}.20 More recently, it has been shown that the PDGFR PTK activity in a rat carotid artery is enhanced by cuff injury to the common carotid artery and is inhibited by a tyrphostin compound.24 Our studies25 as well as other recent studies26 27 28 have assessed the efficacy of different tyrphostin compounds in the inhibition of stenotic and restenotic processes in rat and pig models. The results obtained are controversial because the tyrphostins used in these studies were not strictly specific for major receptor PTKs25 29 or were not effective in the rat model.26 This controversy prompted us to examine the possible antirestenotic efficacy of a highly PDGFR-specific tyrphostin compound, AG-1295,30 31 and to compare its cellular mechanism(s) of action with that of a previously characterized broad-spectrum tyrphostin, AG-17.25 29

In the present study, we evaluated the in vitro effects of a PDGFRß-specific tyrphostin, AG-1295, on the proliferation of rat aortic SMCs in culture and assessed the antirestenotic effects of this tyrphostin in the rat carotid model by a site-specific controlled release delivery system. Furthermore, we provided evidence that in vitro and in vivo effects of AG-1295 are mediated by its inhibitory effects on PDGFRß-triggered tyrosine phosphorylation.


*    Methods
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Materials
Ethylene–vinyl acetate copolymer (EVA, Elvax 40, Du Pont Chemical Co) was washed with water and alcohol to remove impurities.32 AG-17 (3,5-di-tert-butyl-4-hydroxybenzylidene malononitrile) and AG-1295 (6,7-dimethyl-2-phenylquinoxaline) were purchased from Oz Chemicals. Other materials used for drug delivery system preparation were of analytical grade. Cell culture materials and reagents were purchased from Sigma Chemical Co.

Matrix Preparation
AG-1295 and EVA were dissolved in methylene chloride, yielding a 15% (wt/vol) solution as described previously.25 32 The solution was poured into glass Petri dishes and dried, after which the cast polymeric matrix was cut into circular pieces that were 0.2 and 0.6 mm thick and 1.74 cm2 for in vitro release studies and 0.3 mm thick and 1 cmx0.8 cm for implantations in rats. Unidirectional release of the drug was achieved by sealing 1 surface of the matrices with blank EVA film by using a few drops of methylene chloride as glue.

Release Kinetics
For measuring drug release kinetics, 2 circular pieces of sealed AG-1295 matrices with a mean surface area of 1.74 cm2 were separated by aluminum foil and mounted back to back between 2 halves of a diffusion cell, as described previously.25 The cells were filled with polyethylene glycol 300 (PEG 300) and placed in a shaker at 37°C, after which the amount released from each matrix was measured spectrophotometrically at 265 nm in the receiving solution at different time points. PEG 300 was used as the receiving solution instead of physiological buffer because of the extremely low solubility of AG-1295 in water. To keep "sink conditions" (maximum concentration of 10% of saturation), the receiving liquid was replaced at each time point with fresh liquid.

Animals
Animals were obtained from Harlan Laboratories, Jerusalem, Israel, and housed in the animal facilities of the Faculty of Medicine, The Hebrew University of Jerusalem. Experiments conformed to the standards for care and use of laboratory animals of the Hebrew University of Jerusalem. Male rats of the Sabra strain weighing 350 to 420 g were used. The animals were fed standard laboratory chow and tap water ad libitum. All in vivo experiments were performed with the rats under general anesthesia, which was achieved with 80 mg/kg IP ketamine and 5 mg/kg IP xylazine.

Growth Inhibition of Vascular SMCs In Vitro
The cells were isolated and cultured from aortas of adult male Sabra rats by the explant method. After the artery was isolated, the adventitia was peeled off, and the endothelium was removed. The medial tissue was cut into 1x1-mm2 pieces and then adhered to tissue culture dishes. After 1 hour, the growth medium (high glucose DMEM supplemented with 10% FCS, 2 mmol/L L-glutamine, 1000 U/mL penicillin, 100 µg/mL streptomycin, 50 mg/mL gentamycin, and 12.5 U/mL nystatin) was added to the culture dish. Cells were cultured in a humidified atmosphere of 8% CO2/92% air. On reaching confluence (typically after 14 to 16 days), the cells were harvested by use of trypsin/EDTA and passaged with a plating density of 1x104 cells per well (1 mL) in the presence of growth medium. Only passages 1 to 4 were used for these experiments. Stock solutions of AG-1295 were prepared in dimethyl sulfoxide (DMSO) and were diluted in growth medium, yielding concentrations of 5, 10, and 50 µmol/L of AG-1295 and 0.05%, 0.1%, and 0.5% of DMSO, respectively. In control experiments, an equal quantity of DMSO was added to the growth medium. The growth medium (with or without drug) was changed on days 1, 4, 6, 8, and 10. On days 1, 4, 6, and 13 after seeding, the cells were harvested with trypsin/EDTA and counted with a Coulter Counter.

The reversibility of cell growth after AG-1295 washout was assessed in an additional study by omitting drug additions to growth medium after 4 days. The reversion coefficient was calculated by dividing the number of cells growing on day 13 by the number of cells that grew after 6 days.

All cell culture studies were repeated 3 times, and each single experiment was run in triplicate.

In Vitro Viability Assay
SMCs were seeded and grown as described above. Drug or vehicle was added twice on days 1 and 4. On day 6, SMCs were detached from the culture plates by incubation with 0.4 mL trypsin for 5 minutes. Subsequently, cell suspension was diluted with medium and PBS to a total volume of 2.5 mL, and 1 mL of diluted suspension was incubated with 0.2 mL of trypan blue for 10 minutes at room temperature. The percentage of viable SMCs was calculated by means of a hemocytometer. Five squares per count were assessed in a blinded manner.

PTK Inhibition In Vitro Assay
The 10-mL culture dishes were coated with a fibronectin layer (0.5-hour incubation with 5 mL of 5 µg/mL fibronectin in PBS solution [both from Biological Industries] at 37°C). Fibronectin was used to promote adhesion, as suggested previously.33 34 Rat SMCs grown in fibronectin-coated dishes to subconfluence were starved for 48 hours in serum-free medium. AG-1295 (10 µmol/L) in DMSO, AG-17 (10 µmol/L) in DMSO, or vehicle alone was added for 1 hour and then stimulated with 5 ng/mL PDGF-BB or 15% FCS for 10 minutes (in triplicate). Tyrosine phosphorylation was stopped by a wash in ice-cold PBS containing 1 mmol/L sodium vanadate. The cells were scraped from the dishes and lysed in a buffer solution containing 20 mmol/L HEPES, 0.1 mmol/L EDTA, 1 mmol/L PMSF, 10 µg/mL aprotinin, 10 µg/mL soybean trypsin inhibitor, 300 µg/mL benzamidine, 25 mmol/L glycerophosphate, 1 mmol/L sodium orthovanadate, and 0.1% SDS at pH 7.4. Protein content was determined by using a modified Lowry protocol. Equal amounts of protein were separated by SDS-PAGE (7.5% acrylamide gel) and transferred to a polyvinylidene difluoride membrane. The tyrosine-phosphorylated residues were probed with anti-phosphotyrosine antibody (PY-20, No. sc-508, Santa Cruz Biotechnology) and were visualized by an enhanced chemiluminescence (ECL)–based detection system (ECL kit, Amersham).

To demonstrate that indeed the PDGFRß is activated and inhibited, equal amounts of cell lysates were subjected to immunoprecipitation by use of a PDGFRß-specific antiserum (UBI), followed by SDS-PAGE (7.5% acrylamide gel). Immunoblot analysis for phosphotyrosine residues was performed (in triplicate) by using a mix of monoclonal antibodies (4610, UBI; P420, Transduction Laboratories). Visualization of peroxidase-coupled antibodies was achieved by ECL (Amersham).

Rat Carotid Catheter Injury Model
The distal left common and external carotid arteries were exposed through a midline incision in the neck. The left common carotid artery was denuded of endothelium by the intraluminal passage of a 2F balloon catheter (Baxter Healthcare Corp) introduced through the external carotid artery. The catheter was passed 3 times with the balloon distended sufficiently with saline to generate a slight resistance. The catheter was then removed, and the external carotid artery was ligated. A matrix (1 cmx0.8 cm) was wrapped and sutured around the injured portion of the vessel with the drug-releasing surface facing the arterial wall, and the wound was closed with surgical staples. In 16 rats, randomly assigned, 10% AG-1295 matrices (n=8) or plain EVA matrices (n=8) were implanted. The surgical procedure and tyrphostin treatment did not cause mortality or apparent morbidity of the animals. However, 2 explants of the tyrphostin-treated animals were discarded because of technical problems.

All animals were euthanized 14 days after injury by an overdose of pentobarbital. Arteries were perfusion-fixed with 150 mL of 4% formaldehyde solution (pH 7.4) at 100 mm Hg. The right atrium was dissected, and an 18-gauge catheter connected to the perfusion system was inserted in the left ventricle. The arterial segments were dissected, cut, gently separated from the polymer, and postfixed for at least 48 hours in the same fixative solution. The central part of the arterial section wrapped by the matrix (7 to 8 mm) was taken for histological examination. The arterial segments were embedded in paraffin and cut at 8 to 10 sites 600 µm apart, and sections of 6 µm were mounted and stained with Verhoeff’s elastin stain.

Morphometric Analysis
The slides were examined microscopically by an investigator blinded to the type of the experimental group. Six to 8 sections in each slide were evaluated by computerized morphometric analysis (CUE-2 Image Analyzer, Galai Production Ltd), and the averaged section data were further used as a representative of a whole slide for comparisons between groups. The residual lumen, the area bounded by the internal elastic lamina (original lumen), and the area circumscribed by the external elastic lamina ("total arterial area") were measured directly. The degree of neointimal thickening was expressed as the ratio between the area of the neointima and the original lumen (percent stenosis) and as the ratio between the neointimal area to the area of the media. The medial area, an indirect index of SMC viability, was determined as the difference between the total arterial area and the original luminal area.

PTK Inhibition In Vivo Assay
In an additional group of 12 rats, the same balloon injury procedure was performed; this procedure was followed by implantation of AG-1295 10%/EVA (n=6) or plain EVA (n=6) matrices. Intact (n=2) rat carotid arteries served as controls. Three and 14 days after injury, the animals were euthanized (3 animals from each experimental group at each time point). The arterial segments wrapped by the matrices were rapidly retrieved after brief perfusion with cold PBS, rinsed in cold PBS, and immediately deep-frozen (-70°C) until further processing. Frozen rat carotid artery segments were put on dry ice and mechanically minced. Proteins were extracted by using a lysis buffer containing 150 mmol/L NaCl, 50 mmol/L Tris-HCl, 1% Triton X-100, 10 mmol/L EDTA, 1 mmol/L PMSF, 100 µmol/L sodium orthovanadate, and 1% aprotinin. Protein content was determined by using a modified Lowry protocol. Protein samples (40 µg each) were subjected to SDS-PAGE (7.5%) and blotted onto a nitrocellulose membrane (Hybond C Extra, Amersham). Tyrosine-phosphorylated proteins were detected by immunoblotting with horseradish peroxidase–conjugated anti-phosphotyrosine antibody RC2OH (Transduction Laboratories), followed by the application of ECL (Amersham) and autoradiography. Similarly, detection of the receptor protein was performed by immunoblotting with the IgG fraction of a polyclonal PDGFRß-specific antiserum (UBI); this procedure was followed by several washing steps and the application of a horseradish peroxidase–conjugated donkey anti-rabbit antibody (Amersham). Visualization was achieved by chemoluminescence and autoradiography. The receptor blot and the phosphotyrosine blot were produced in parallel under exactly the same conditions (in duplicate).

Quantification of the PDGFRß band was made after Western blot analysis by use of the software packages of ImageReader LAS and ImageGauge (Fuji). Band intensity was normalized to the control level, termed 100%.

Drug Release In Vivo
To determine the drug release kinetics in vivo, 0.5% and 10% AG-1295 matrices were implanted subcutaneously in 12 rats. In preliminary experiments, the release rate from subcutaneously implanted devices was found to be the same as from perivascularly implanted matrices (data not shown). The polymers were retrieved 1, 7, 15, and 28 days after implantation, and the amount of AG-1295 released was determined by the difference between initial and residual drug content in the matrices. This was achieved by spectrophotometric analysis of the drug in retrieved matrices compared with unimplanted matrices after dissolution in methylene chloride.

AG-1295 Disposition After Perivascular Delivery
The uptake of AG-1295 by arterial tissue and plasma drug concentrations after perivascular delivery were assessed in the rat carotid model by high-performance liquid chromatography (HPLC) assay. Balloon injury and matrix implantation were performed as described above. Animals were euthanized 1 and 14 days after balloon injury and perivascular matrix implantation. Arterial segments underlying the delivery system were explanted, dissected free from perivascular tissue, lyophilized, and dry-weighed. The small size of the explanted tissues necessitated analyzing a pool of carotid arteries from 4 animals at each time point. Arterial tissue was homogenized, and the drug was extracted with chloroform. The chloroform was evaporated under vacuum, and the dry residue was resuspended in 100 µL of the mobile phase (70% acetonitrile and 30% water). Twenty microliters of the final solution was injected into an HPLC system (Kontron Instruments) with a chromatography column (RP-18, Merck). The drug was detected at 265 nm by using AG-1296, a dimethoxy derivative of AG-1295, as an internal standard. The experiments were performed by an investigator blinded to the type of the experimental group.

Statistics
Data are expressed as mean±SEM. Differences between groups in experiments assessing antirestenotic activity of AG-1295 were termed statistically significant by the unpaired Student t test at P<0.05 and by ANOVA with the Fisher post hoc test.


*    Results
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Growth Inhibition of SMCs in Culture
The addition of 5, 10, and 50 µmol/L AG-1295 to cultures of SMCs explanted from rat aortas resulted in a significant dose-dependent growth inhibition of the SMCs compared with vehicle-treated control cells (Figure 1ADown). Growth-inhibited SMCs regained their normal growth pattern after tyrphostin washout, indicating the absence of long-term effects/toxicity of AG-1295. The reversion coefficients (cells growing on day 13÷cells growing on day 6) were 3.63, 3.74, and 2.75 for 5, 10, and 50 µmol/L AG-1295–treated cells, respectively (Figure 1BDown). The growth coefficient of control samples for the same time period was 2.9.



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  		Figure 1. A, Growth curves of rat aortic SMCs treated with 5, 10, and 50 µmol/L AG-1295 in growth medium containing 0.05%, 0.1%, and 0.5% DMSO, respectively, after stimulation with 10% FCS. Growth medium containing DMSO at respective concentrations (with or without drug) was replenished on days 1, 4, 6, 8, and 10 in all experiments. The cells were harvested on days 4, 6, and 13 for counting. Data are mean±SD (in some data points, the SD is too small to be shown). B, Growth curves of rat aortic SMCs untreated (control) and treated with 5, 10, and 50 µmol/L AG-1295 after stimulation with 10% FCS. AG-1295 treatment was on days 1 and 4 or days 1, 4, 6, 8, and 10, as indicated. The dotted lines diverging from the solid lines on day 6 represent cell growth after treatment was withdrawn (see text for reversion coefficients). Data are mean±SD (in some data points, the SD is too small to be shown).

Drug Delivery System of AG-1295
Some factors affecting the release rate of the drug have been determined to modulate drug release according to the animal species and response. The extremely low solubility of AG-1295 in simulated physiological buffer (PBS) and in water precluded release kinetics studies in these fluids, so they were performed in PEG 300. For assessing the impact of matrix thickness and drug load on the release profile of AG-1295, 3 types of matrices were compared on a paired basis. All matrices irrespective of thickness and drug load released the drug in an exponential manner, ie, decreasing release rate over time. The release rate from 10% matrices was somewhat slower than that obtained from 0.5% matrices with the same thickness (0.6 mm). Similarly, the release rate from the thicker matrices (0.6 mm) was slower than that obtained from the thinner matrices (0.2 mm) with the same drug content (10%). As seen in Figure 2Down, 37%, 46%, and 52% of the incorporated drug was released after a 1-week implantation from 10% matrices that were 0.2 mm thick, 10% matrices that were 0.6 mm thick, and 0.5% matrices that were 0.6 mm thick, respectively. Approximately 98% of the drug was released after 28 days from the 0.2-mm-thick 10% matrices, and {approx}90% of the drug was released from 0.6-mm-thick matrices (both 0.5% and 10%). This release rate corresponds to an average dose of 2 and 40 µg/kg per day for 0.5% and 10% matrices, respectively. It should be noted that the "burst effect" (rapid release rate at the beginning), present in the release curves in vitro, was not evident with the subdermal implants.



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  		Figure 2. Release profile of AG-1295 from EVA matrices implanted subdermally in rats compared with release profile in vitro (PEG-300).

Cell Viability Assay
The trypan blue exclusion assay showed that the number of viable cells did not significantly differ between the groups consisting of growth medium with no additions, growth medium with 0.5% DMSO, and 50 µmol/L of AG-1295 in growth medium containing 0.5% DMSO (90.7±1.8%, 86.6±3.5%, and 83.6±1.3%, respectively).

Inhibition of Tyrosine Phosphorylation by AG-1295 In Vitro
The proposed mechanism of AG-1295 antiproliferative action is an inhibition of PDGF-triggered tyrosine phosphorylation cascades in SMCs. The degree of tyrosine phosphorylation was assessed by Western blot with phosphotyrosine-specific antibody in SMC extracts. As seen in Figure 3ADown, several bands characteristic of PDGF–BB–stimulated or FCS-stimulated SMCs disappeared in the presence of AG-1295. It is noteworthy that the band representing PDGFR ({approx}190 kDa) existed in lanes corresponding to AG-17 treatment but was virtually absent in the AG-1295–treated cells. The specificity of the above-mentioned band was validated by immunoprecipitation of PDGFRß with the specific antibody before probing with anti-phosphotyrosine antibody (Figure 3BDown). This inhibition of tyrosine phosphorylation of PDGFRß was reversible, in view of the fact that the band completely reappeared after drug washout (Figure 3CDown, lanes 4 and 6).



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  		Figure 3. A, Effects of 10 µmol/L AG-17 and 10 µmol/L AG-1295 pretreatment on tyrosine phosphorylation induced by 5 ng/mL PDGF-BB or by 15% FCS. Shown is a Western blot of whole-cell lysates probed with anti-phosphotyrosine antibody and detected by ECL. On the left of lane 1 are the molecular mass markers. Lanes are as follows: lane 1, unstimulated SMC; lane 2, SMC stimulated by 5 ng/mL PDGF-BB; lane 3, SMC stimulated by 15% FCS; lane 4, SMC stimulated by 5 ng/mL PDGF-BB pretreated by 10 µmol/L AG-17; lane 5, SMC stimulated by 15% FCS pretreated by 10 µmol/L AG-17; lane 6, SMC stimulated by 5 ng/mL PDGF-BB pretreated by 10 µmol/L AG-1295; and lane 7, SMC stimulated by 15% FCS pretreated by 10 µmol/L AG-1295. B, Effect of AG-1295 pretreatment on PDGF-BB–induced tyrosine phosphorylation of PDGFRß. Cell lysates were subjected to immunoprecipitation by use of a PDGFRß-specific antiserum, followed by SDS-PAGE and immunoblot analysis for phosphotyrosine residues with use of a mix of monoclonal antibodies. Visualization of peroxidase-coupled antibodies was achieved by using ECL. On the left of lane 1 are the molecular mass markers. Lanes are as follows: lane 1, unstimulated SMC; lane 2, unstimulated SMC pretreated by 10 µmol/L AG-1295; lane 3, SMC stimulated by 10 ng/mL PDGF-BB; lane 4, SMC stimulated by 10 ng/mL PDGF-BB pretreated by 10 µmol/L AG-1295; lane 5, SMC stimulated by 100 ng/mL PDGF-BB; and lane 6, SMC stimulated by 100 ng/mL PDGF-BB pretreated by 10 µmol/L AG-1295. C, Reversibility of AG-1295–induced inhibition of tyrosine phosphorylation 16 hours after tyrphostin washout and addition of fresh growth medium. Shown is a Western blot of whole-cell lysates probed with anti-phosphotyrosine antibody (PY-20) before and 16 hours after tyrphostin washout. ECL was used for detection. On the left of lane 1 are the molecular mass markers. Lanes are as follows: lane 1, unstimulated SMC; lane 2, SMC stimulated by 5 ng/mL PDGF-BB; lane 3, SMC stimulated by FCS; lane 4, SMC stimulated by 5 ng/mL PDGF-BB pretreated by 10 µmol/L AG-1295; lane 5, SMC stimulated by FCS pretreated by 10 µmol/L AG-1295; lane 6, SMC stimulated by 5 ng/mL PDGF-BB pretreated by 10 µmol/L AG-1295 at 18 hours after AG-1295 washout; lane 7, SMC stimulated by FCS pretreated by 10 µmol/L AG-1295, 18 hours after FCS washout.

AG-1295 Retention in Arterial Tissue After Local Delivery
The experiments assessing drug content at the delivery site were conducted by using pooled rat arterial tissues to allow detection of drug uptake. The mean AG-1295 concentration in arterial segments underlying delivery devices 1 and 14 days after matrix implantation was 741 and 21.1 ng/mg dry tissue, whereas blood AG-1295 concentration was below the HPLC detection limit of 1 ng/mL.

Effects of AG-1295 on Neointimal Formation in Rats
The extent of mean neointimal formation 14 days after implantation of AG-1295 matrices (n=6), measured as a percent of luminal stenosis, was 19.75±3.80% compared with 30.56±3.12% in the control group (n=8, P<0.05; Figure 4Down). Similarly, statistically significant reductions of the mean neointima-to-media ratio, 0.65±0.12 and 0.99±0.08, were found in AG-1295–treated and control groups, respectively (P<0.05).



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  		Figure 4. Inhibition of neointimal formation 14 days after balloon injury to rat carotid arteries by perivascular implantation of 10% AG-1295/EVA matrices (1x0.8 mm, 3 mm thick). A, There is a significant difference in the percentage of luminal stenosis and the mean neointima-to-media area ratio between AG-1295–treated (n=6) and control (n=8) groups. B, Photomicrographs of representative histological sections (van Giesen’s elastic stain, magnification x320) are shown. On the left is a section of rat carotid artery locally treated by blank EVA matrices. On the right is a section of rat carotid artery locally treated by 10% AG-1295 implants.

The mean area of the media in control and AG-1295–treated groups did not differ significantly (0.095±0.006 mm2 and 0.089±0.009 mm2, respectively; P>0.2; Figure 4Up).

Effects of AG-1295 Treatment on Tyrosine Phosphorylation and PDGFRß Upregulation In Vivo
PDGFRß expression was enhanced to 133% and 148% compared with preinjury levels (100%) at 3 and 14 days after injury (Figure 5ADown; compare lanes 1 versus 2 and 3). Local AG-1295 treatment almost completely inhibited PDGFRß expression 3 days after injury, from 133% to 11% (Figure 5ADown; compare lanes 2 and 4). Two weeks after injury, the receptor expression was partially recovered in tyrphostin-treated arteries (46%), but the level of PDGFRß was still significantly lower than that observed in the corresponding control group (148%, Figure 5ADown; compare lanes 3 and 5).



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  		Figure 5. Immunoblot analysis of the PDGFRß protein (A) and phosphotyrosine-containing proteins (B). Extracted proteins (40 mg) were subjected to SDS-PAGE followed by immunoblot analysis of PDGFRß (arrow) by use of a polyclonal antiserum (A) and analysis of tyrosine phosphorylation of PDGFRß protein (arrowheads) with use of RC2OH antibodies (B). Detection of hybridizing antibodies was obtained by ECL. Samples were derived from 5 different animal groups, reflecting 5 different protocols: 1, intact control group (no injury, no matrix; n=2); 2, control group short-term (injury, plain matrix for 3 days; n=3); 3, control group long-term (injury, plain matrix for 2 weeks; n=3); 4, treatment group short-term (injury, 10% AG-1295 matrix for 3 days; n=3); and 5, treatment group long-term (injury, 10% AG-1295 matrix for 2 weeks; n=3).

A change in tyrosine phosphorylation pattern of multiple bands was observed 3 days after injury (Figure 5BUp; compare lanes 1 and 2). Some bands (such as 190 kDa, 120 kDa, 97 kDa, and 42 to 44 kDa) were enhanced, and some were decreased (such as the one at 70 kDa), whereas some others remained unchanged (such as <66 kDa). Phosphotyrosine levels (Figure 5BUp, lane 3) were decreased 14 days after injury compared with basal levels and levels 3 days after injury (Figure 5BUp, lanes 1 and 2, respectively). The intensities of the 190-kDa band were 134% and 42% at 3 and 14 days after injury (lanes 2 and 3) compared with the control level (100%, lane 1).

Notably, 3- and 14-day treatments decreased the degree of tyrosine phosphorylation in SMCs below basal levels (Figure 5BUp; compare lanes 1, 4, and 5). Moreover, the band corresponding to a protein of PDGFRß molecular weight (Figure 5BUp, arrowheads), which also exhibited upregulation (134%) 3 days after injury (Figure 5BUp, lane 2), was significantly diminished (to 6%) by AG-1295 treatment (Figure 5BUp, arrowheads, lane 4). The intensity of the 190-kDa band 14 days after injury was further decreased from 42% to 2% (Figure 5BUp, arrowheads, lanes 3 and 5).


*    Discussion
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 *Discussion
down arrowReferences
 
The mechanism of SMC proliferation due to serum stimulation is thought to involve the dimerization of growth factor receptors, which triggers multiple phosphorylation of specific tyrosine residues in receptor molecules.35 36 This autophosphorylation process initiates a chain of signaling events leading to transfer of the initial signal to the cell nucleus, to a shift in transcription pattern, and eventually to proliferation.37 The mechanism of action of AG-1295 is most probably the same as was shown for the dimethoxy derivative, AG-1296, another specific inhibitor of PDGFRß.31 Tyrphostin, an ATP-competitive inhibitor of the receptor kinase, intervenes in the first step of the signaling cascade and abolishes PDGF autophosphorylation, as shown in the present study. It is important to note that inhibition of SMC proliferation due to AG-1295 pretreatment was reversible. When AG-1295 was washed out and fresh medium without drug was added, growth-inhibited cultured SMCs regained a normal proliferation pattern, indicating no long-term effect/toxicity of the drug. Moreover, trypan blue exclusion assay of cell viability showed no significant augmentation in cell death in AG-1295–treated cells.

In subconfluent SMC monolayers, we studied upregulation of tyrosine phosphorylation after stimulation by PDGF-BB and FCS (Figure 3AUp). Marked enhancement of multiple bands can be seen 10 minutes after a challenge with PDGF-BB or FCS (Figure 3AUp). This upregulation was effectively blocked by pretreatment with AG-1295 and, to a markedly lesser extent, by AG-17, a nonspecific PTK inhibitor (Figure 3AUp). A similar observation of PDGF-BB–promoted tyrosine phosphorylation in rat cultured SMCs was recently reported by Majack et al.38 The interpretation of the nature of the band at 190 kDa that manifested upregulation of tyrosine phosphorylation as PDGFRß was facilitated by the immunoprecipitation of PDGFRß before Western blotting with anti-phosphotyrosine antibody (Figure 3BUp). Other important proteins demonstrating an increase in phosphotyrosine content after PDGF-BB or FCS treatment in the present study could be (1) the focal adhesion kinase (FAK, 125 kDa), (2) the regulatory subunit of phosphatidylinositol-3' kinase (85 kDa), (3) the c-Src (60 kDa), or (4) the mitogen-activated protein (MAP) kinase isoforms (42/44 kDa). FAK,39 phosphatidylinositol-3' kinase,40 and MAP kinase (ERK 1/2)41 were reported to be rapidly phosphorylated on distinct tyrosine residues on PDGF-BB stimulation. The inhibitory effect of AG-1295 on PDGF-BB–induced tyrosine phosphorylation of PDGFRß was reversible, in view of the fact that complete recovery was observed after washout of the inhibitor (Figure 3CUp). This important finding corresponds with the observed recovery of normal growth pattern in SMCs after switching to AG-1295–free medium and with the results of the viability study, indicating nontoxic long-term effects on SMCs.

The changes in the level of PDGF A- and B-chain expression take place as early as 4 hours after balloon injury in the rat carotid model42 and continue for at least 2 weeks, well after the wave of medial proliferation and the peak of SMC migration to neointima abate. Thus, any therapeutic approach designed to lessen the PDGF-mediated contribution to neointimal growth should take into account the need for continuous inhibition of an aberrantly activated PDGF/PDGFR system. The different effects of short- and long-term inhibition of another downstream regulator of SMC proliferation, c-myc, on neointimal formation in rats were recently reported by Edelman et al.43 In that study, the sustained local delivery of c-myc antisense construct from perivascularly implanted devices was shown to reduce neointimal formation, whereas the short-term delivery was proven to be ineffective. The use of local drug delivery can minimize problems such as the inability to use pharmacologically active doses because of side effects and systemic inactivity that is due to rapid metabolism and/or hydrolysis before reaching the target. Indeed, AG-1295 is rapidly degraded after intramuscular or intravenous administration (data not shown), whereas the hydrophobic drug delivery system maintains drug stability.25 29 Although impractical in the setting of percutaneous transluminal coronary angioplasty, perivascular delivery is a good choice for elucidation of the pharmacodynamic mechanisms of drug action because of its relative technical simplicity and ability to modulate drug release. Moreover, adventitial delivery might be of therapeutic value in the setting of vascular grafts. Nevertheless, more practical delivery approaches should be adopted in view of future clinical implications. In this context, we recently reported on the marked antirestenotic activity of AG-1295–loaded polymeric nanoparticles administered intraluminally in the pig carotid model.44

A drug delivery system with a protracted release was formulated. No burst effect was found in vivo, probably because of the slower rate of fluid exchange and the lack of sink conditions in the subdermal milieu. The exponential AG-1295 release kinetics from EVA-based matrices in vitro were found to conform to the half-time relation, and a good correlation was found between the amount of drug released and half-time for the various drug loads and matrix thickness45 (graph not shown). The release kinetics in vitro performed with PEG 300 approximated the in vivo release pattern. The determination of the drug release kinetics permits the rational design of a suitable drug delivery system with respect to geometrical size, delivery rate, and duration, which is required in larger animals. For example, porcine carotid implants, which require a scale of up to {approx}3 times the delivery rate, could be easily achieved by using a larger surface area matrix to cover the larger injured area and by increasing the thickness of the matrix to enable adjustment to the desired delivery rate (see Figure 2Up). Only the diameter of the artery should be considered, because only a local effect is sought; the size of the animal need not be considered.

An average AG-1295 content of 711 and 29.1 ng/mg dry tissue was found in arterial segments underlying the treated site, whereas plasma drug concentration was below the detection limit. Given the high specificity of AG-1295 to the PDGFRß PTK shown here and earlier19 30 and the marked antirestenotic effect observed in the rat carotid model (Figure 4Up), this concentration was sufficient to inhibit PDGF-induced activation of SMCs in injured arteries. The recently reported low residence time of heparin in arteries after perivascular delivery46 was attributed to a rapid distal washout of hydrophilic heparin. In contrast, a highly hydrophobic compound such as AG-1295 should exhibit an extended residence time in the vessel wall.

We previously reported a marked antirestenotic activity of another tyrphostin compound, AG-17, in the same rat model.25 Although as effective as AG-1295 treatment, the local delivery of AG-17 from perivascularly implanted matrices was associated with certain signs of medial toxicity. The inhibitory effect of AG-17 on pig44 and rat47 SMCs is not reversible, and SMCs do not resume proliferation even at a low tyrphostin concentration of 1 µmol/L. Moreover, PDGF-BB–induced tyrosine phosphorylation of PDGFRß is effectively blocked by pretreatment with AG-1295 but not by AG-17 (Figures 3AUp and 3CUp). These findings are in accord with the low specificity of AG-17 to the PDGFRß, because its mechanism of action is also related to blocking GTP-using enzymes48 and mitochondrial functions.49 In contrast, the local delivery of AG-1295, a specific PDGFR inhibitor, was not associated with toxic reactions. The medial areas and the overall histological appearance of arterial segments in the treatment group were the same as in the control group.

An immunoprecipitation of PDGFRß from rat arterial tissue (similar to the one performed on cell lysates, Figure 3BUp) was not performed because it is very difficult to immunoprecipitate a certain receptor tyrosine kinase from the whole-tissue lysates and because of the large number of animals required to pool enough material. Nevertheless, several lines of evidence indicate that the protein migrating at 190 kDa is PDGFRß. The same molecular mass and the disappearance of the tyrosine-phosphorylated band at 190 kDa in the presence of AG1295 indicate the identity of the protein at 190 kDa. Moreover, it was shown recently (M. Kovalenko, F.D. Böhmer, personal communication, 1999) that only PDGFRß could be isolated from fibroblast lysates by affinity binding to AG-1295 and that there is no other molecule expressed in mesenchymal cells that directly binds to AG1295 with high affinity.

We hypothesized that the beneficial effect of AG-1295 treatment on neointimal formation is mediated by PDGFRß PTK inhibition of SMCs. We,25 as well as others,24 40 have previously shown that balloon injury upregulates overall tyrosine phosphorylation in vascular SMCs. In accordance with these early findings, we have shown in the present study (Figure 5BUp) that tyrosine phosphorylation of PDGFRß (190 kDa) and of several other proteins was increased. The latter proteins migrating at sizes of 125 kDa and 42/44 kDa and disappearing under AG-1295 treatment most likely represent downstream mediators of the PDGFRß, such as p125FAK and MAP kinase isoforms. After 14 days, the protein of the receptor remains upregulated (Figure 5AUp, arrowhead, lane 3), but activation was decreased to 42% (Figure 5BUp, arrowheads, lane 3 versus lane 1). One possible explanation of the general decrease of the phosphorylation levels is a dephosphorylation that might be a specific response to PDGFR activation (as in the case of the 70-kDa band). Another explanation is that reduced phosphorylation in lane 3 (Figure 5BUp) is due to the accumulation of extracellular matrix in the injured vessels. Because the blot is normalized for protein loading, the SMC-derived proteins become diluted, resulting in weaker bands.

Majesky et al42 have shown that the expression of PDGFRß mRNA was downregulated immediately after injury and reached {approx}20% of basal level 4 hours after ballooning. Receptor expression was then steadily restored and at 1 week after injury exceeded by 2 times the basal level of expression. These results were confirmed recently by Sirois et al,50 who showed that balloon injury enhanced PDGFRß expression in SMCs of the media and nascent neointima after 2 weeks. Panek et al40 have shown a very weak basal expression of PDGFRß on SMCs of intact rat carotid arteries that was not significantly changed 2 days after injury. The respective band was markedly enhanced in blots from arteries of rats euthanized 7 to 12 days after injury and then decreased at later time points. All of these results correspond well with our finding of moderately upregulated PDGFRß expression on day 3 after injury and a marked increase in receptor expression after 2 weeks (Figure 5AUp). Our preliminary experiments after immunoprecipitation of PDGFRß validated that the band migrating at 190 kDa is PDGFRß, as was shown by Panek et al40 and by the findings in the present study (190 kDa and the similar band location after immunoblotting with anti-PDGFRß and anti-phosphotyrosine antibodies; compare Figure 5AUp and 5BUp).

The PDGFRß expression that was enhanced 3 days after injury was almost completely inhibited by the local AG-1295 treatment (Figure 5AUp; compare lanes 2 and 4). Two weeks after injury, the receptor expression was partially recovered in tyrphostin-treated arteries, but the level of PDGFRß was still significantly lower than that observed in the control group (Figure 5AUp; compare lanes 3 and 5).

The band corresponding to PDGFRß activation that exhibited a marked enhancement after injury was significantly reduced in both treatment groups (Figure 5BUp, arrowheads, lanes 2 and 3 versus lanes 4 and 5, respectively). Treatments at 3 and 14 days decreased the degree of tyrosine phosphorylation below basal levels (Figure 5BUp, lane 1 versus lanes 4 and 5, respectively). Because there is a baseline activation of the receptor by PDGF for maintenance of normal vascular homeostasis, the PDGFRß tyrosine phosphorylation is reduced below baseline levels after AG-1295 treatment, and some of the other bands disappear as well. The disappearance of the other bands after AG-1295 treatment could be dependent, at least in part, on phosphorylation of these proteins via PDGFRß activation. In the absence of PDGF activity, they are no longer activated.

The formulation of the specific PDGFRß inhibitor, AG-1295, in our delivery system resulted in a release pattern that adequately matched the PDGFRß expression time course. Controlled and extended release of tyrphostin from the matrices virtually abolished PDGFRß expression on day 3 after injury and markedly inhibited the expression 14 days after injury.

In conclusion, a significant antistenotic effect of the PDGFRß PTK–specific tyrphostin compound, AG-1295, was observed in the rat carotid model. This effect paralleled the marked antiproliferative activity of AG-1295 to the rat vascular SMCs in culture monolayers. In vitro and in vivo effects were mediated by reversible inhibition of tyrosine phosphorylation upregulation. In addition to reduction of PDGFRß autophosphorylation, tyrphostin treatment led to downregulation of receptor expression in the arterial wall after balloon injury. Because PDGFRß-mediated signaling has been proven to play an important role in postangioplasty response in nonrodent mammals10 27 28 and humans,51 52 its specific inhibition represents a new and interesting approach that warrants further investigation.


*    Acknowledgments
 
This study was supported in part by a grant from the Joint German-Israeli Research Project (BMBF and MOSA) and The David R. Bloom Center for Pharmacy. G.G. is affiliated with the David R. Bloom Center for Pharmacy at The Hebrew University of Jerusalem. J.W. is supported in part by a grant from the Land Baden-Württemberg (Landesforschungsschwerpunkt).


*    Footnotes
 
1 Both authors contributed equally to this study. Back

Received January 7, 1999; accepted September 17, 1999.


*    References
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