Platelet-Derived Growth Factor Receptor Antagonist STI571 (Imatinib Mesylate) Inhibits Human Vascular Smooth Muscle Proliferatio

(A) Inhibition of human coronary artery smooth muscle cells (hCASMC) proliferation by increasing concentrations of imatinib. hCASMC were cultured with imatinib and serum or with serum alone (positive control), and cells were counted for inhibition of cell
Viability of human coronary artery smooth muscle cells (A) or human coronary artery endothelial cells (B). Viability was assessed by trypan blue exclusion. There was no decrease in hCASMC viability with increasing concentrations of imatinib except at the
(A) Imatinib inhibits human coronary artery smooth muscle cell migration (hCASMC) in a dose-dependent manner. Human CASMC were placed on the topside of an 8 µm pore Transwell filter. Chemotaxis to 20 ng/ml PDGF-BB and 1%BSA was determined after 24 hours i
 Effect of imatinib on PDGF-BB and FBS-induced tyrosine phosphorylation of PDGF-b receptor in hCASMCs. Inset measures the  IC50 of imatinib. Error bars are SEM.
Percent restenosis measured by quantitative coronary angiography 28 days after balloon injury and stent implantation. Error bars are SEM.
There was no difference in the neointima between any of the groups. Neotimimal area was the average area of three separate cross-sections of the stented artery. Error bars are SEM.
Author(s): 

Timothy A. Hacker, PhD, Michael O. Griffin, PhD, Brian Guttormsen, MD, Scott Stoker, BS, Matthew R. Wolff, MD

Polymer-coated stents eluting either rapamycin or paclitaxel, drugs that prevent entry of VSMCs into the proliferative cell cycle and inhibit neointimal formation following arterial injury, have dramatically decreased the risk of restenosis following coronary stent implantation.1 However, recent studies suggest that rapamycin- and paclitaxel-eluting stents are associated with a higher risk of delayed or late stent thrombosis compared to bare-metal stents.2,3 These observations, as well as the possibility that resultant myocardial infarction associated with late stent thrombosis may be associated with increased mortality relative to the use of bare-metal coronary stents,4 have lead to the convening of an unprecedented review by the U.S. Food and Drug Administration Circulatory System Devices Panel on the safety of drug-eluting stents (DES). While the impact of late stent thrombosis on mortality following DES implantation relative to bare-metal stents remains controversial, an AHA/ACC/SCAI/ACS/ADA Task Force would later publish a scientific advisory regarding antiplatelet therapy following drug-eluting coronary stenting.5
Delayed or late stent thrombosis appears to be associated with delayed re-endothelialization of the stented coronary segments in human autopsy specimens.6 Both rapamycin and paclitaxel have been shown to inhibit vascular endothelial cell proliferation in vitro.7,8 An ideal pharmacologic agent eluted from a stent to prevent restenosis would inhibit critical signal transduction pathways involved in VSMC proliferation and migration without affecting vascular endothelial cell proliferation. The platelet-derived growth factor (PDGF) signal transduction pathway, initiated by PDGF binding to its protein tyrosine kinase receptor (PDGF-R), is potentially one candidate pathway. Inhibition of PDGF signal transduction has been shown to reduce neointimal proliferation in animal models of arterial injury.9–13 Imatinib mesylate is a selective inhibitor of several protein receptor tyrosine kinases including the chimeric BCR-ABL fusion oncoprotein responsible for chronic myeloid leukemia14 and the c-KIT receptor activated in gastrointestinal stromal tumors.15 Previous studies also indicate that imatinib inhibits PDGF receptors in the nanomolar concentration range, but has little effect on VEGF receptors at 1,000-fold higher concentrations.16 These data suggest that imatinib might be an ideal active pharmacologic agent for drug-eluting coronary stents due to the selective inhibition of neointimal proliferation without negative effects on endothelial cell proliferation.
Our aim in this study was to evaluate the effects of imatinib on PDGF-stimulated human coronary VSMCs and vascular endothelial growth factor (VEGF)-stimulated human coronary endothelial cells in culture and in an in vivo swine coronary artery injury model. We hypothesized that imatinib would selectively inhibit human coronary VSMC migration and proliferation without affecting coronary endothelial cells in vitro. We also hypothesized that imatinib-loaded DES would inhibit neointimal proliferation in a standard porcine model of in-stent restenosis.

Methods

Cell culture. Human coronary artery smooth muscle cells (hCASMC, Clonetics Corp., San Diego, California) were maintained in smooth muscle growth medium-2 (SmGM-2, Clonetics) containing 5% fetal bovine serum (FBS), while human coronary artery vascular smooth muscle cells (hCAEC, Clonetics) were maintained in microvascular endothelial growth medium also containing 5% FBS (EGM-MV, Clonetics). hCASMC assays were performed in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) heat-inactivated FBS, 0.25 µg/ml amphotercin B, and 50 µg/ml gentamycin (assay media), while hCAEC assays were performed using EGM-MV. Throughout the course of the experiments, cells from the fourth through sixth passages were used. Reagents. Imatinib was a gift from Norvartis Pharma AG (Basel, Switzerland). Recombinant human PDGF-BB and VEGF were obtained from Cal Biochem (San Diego, California). Antiphosphorylated PDGFR-b was obtained from Santa Cruz Biotech (Santa Cruz, California).
Proliferation assays. hCASMCs were seeded in assay media in 24-well tissue culture-treated plates at an initial density of 2,500 cells per well (2 cm2). Upon reaching subconfluence (75–80%), the cells were serum-starved for 48 hours in serum-free assay media. The medium was then changed to assay medium containing imatinib or vehicle and incubated for an additional 48 hours. Cells were then trypsinized for manual counting on a hemacytometer. Four wells were counted, each in quadruplicate, and averaged for each experiment. Proliferation values were normalized to uninhibited control wells (positive control; assay media with no imatinib) after subtracting unstimulated wells (negative control; assay medium minus FBS). hCAECs were seeded at 5,000 cells/well in assay media in 24-well tissue culture-treated plates. Upon reaching subconfluence (75–80%), cells were serum-starved for 48 hours in serum-free EGM-MV, and then treated with EGM-MV containing various concentrations of imatinib. The cells were allowed to grow for another 48 hours and then subjected to manual counting as detailed above.
Viability. Cell viability was assessed via trypan blue exclusion.
Migration. Cell migration was quantitated using Transwell culture chambers (Costar, Cambridge, Massachusetts) consisting of upper and lower culture chambers separated by a filter insert with 8.0 µM pores. A total of 4,000 cells (hCASMC and hCAEC) were seeded in the upper chamber in assay media containing varying concentrations of imatinib or vehicle, and stimulated with 20 ng/ml PDGF-BB (hCASMC) or 20 ng/ml VEGF (hCAEC), respectively. Cells remaining on the top side of the filter were scraped off and the cells that migrated through to the bottom side were fixed with ice-cold methanol. Nuclei were visualized by staining with Harris’s hematoxylin stain and counted in 8 high-power (400x) fields per filter. Assays were performed in quadruplicate in 4 independent experiments. Migration values were normalized to uninhibited control wells (positive control; supplemented media with no imatinib) after subtracting unstimulated wells (negative control; supplemented media without PDGF-BB or VEGF and imatinib).
Autophosphorylation. hCASMC were seeded in a 100 mm culture dish at an initial density of approximately 5 x 105 per dish in assay media. Subconfluent cultures (75–80%) were serum-starved for 48 hours and then switched back to assay media alone or assay media containing increasing concentrations of imatinib for 60 minutes prior to activation. Cells were activated by the direct addition of 50 ng/ml PDGF-BB and further incubated for 5 minutes. After the 5-minute activation, cells were washed with ice-cold PBS buffer, lysed for 15 minutes at 4ºC, scraped and transferred to a 1.5 ml Eppendorf tube. Cells were lysed for an additional 90 minutes with constant agitation at 4ºC (lysis buffer TBS pH 7.0; 10 mg/ml PMSF; 100mM Na3VO4; 10 mg/ml leupeptin; 100mM Aprotonin; 1% DOC). Cell lysates were solubilized in 2X SDS-PAGE sample buffer and boiled for 5 minutes. Proteins were resolved by 8% SDS-PAGE, transferred to membrane, and detected using the enhanced chemiluminescence western blotting detection system, probing with anti-pPDGFR- (Santa Cruz Biotechnology) at 1 µg/ml and an appropriate horseradish peroxidase-conjugated secondary antibody.
Neointimal proliferation in a porcine coronary stent model. Eight pigs (4 male) weighing 29.8 ± 4.7 kg were pretreated with oral aspirin (325 mg qd) and clopidogrel (75 mg qd) for 3 days prior to the initial surgery. Animals were maintained on isoflurane (0.8–3.0%, inhalation) for the duration of the procedure as approved by the institutional animal protocol review board and in accordance with the Guide for the Care and Use of Laboratory Animals. An 8 Fr sheath was introduced into the right carotid artery after intravenous heparin administration. A baseline angiogram of the left coronary system was obtained with a 7 Fr AL2 guide catheter. Initimal injury in the mid-left anterior descending coronary artery was created by a 30-second inflation of a slightly oversized angioplasty balloon (1.2:1 balloon-to-artery diameter ratio). Following oversized predilatation, an appropriate-sized stent (1:1 stent-to-artery diameter ratio) was deployed. This protocol was repeated for the left circumflex coronary artery using the AL 2 guide and the right coronary artery using a 7 Fr JR 4 guide catheter. The stents (Invastent, Invatec, Roncadelle, Italy) (bare-metal, polymer-coated only, or polymer + imatinib mesylate, 600 µg/stent) were randomly assigned to each artery. All the hardware was removed and the carotid artery, muscle layers and skin were repaired and the pig recovered. Treatment with oral aspirin (325 mg qd) and clopidogrel (75 mg qd) was continued for the 28-day follow-up period. Twenty-eight days after stent implantation, angiograms were performed for each coronary artery as described above. Quantitative coronary angiography (QCA) was performed (Camtronics, Inc., Hartland, Wisconsin). The animals were euthanized without awakening from anesthesia, and the heart was perfused through the aortic root with saline until the blood was cleared, and then for 10 minutes at 100 mmHg with buffered formalin. The stented arteries were excised for histopathologic examination as described previously.17 All investigators were blinded to the treatment.
Statistical analysis. All data are presented as mean ± standard deviation. Significant differences between experimental groups were determined by one-way ANOVA. Differences among experimental groups were considered to be statistically significant when p < 0.05.


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