ABSTRACT: Background. Cell-specific inhibition of vascular smooth muscle cells, the primary constituent of neointima following arterial injury, without deleterious effects on vascular endothelial cell function may be a critical requirement for drug-eluting stents that are not prone to excess late thrombosis. We hypothesized that imatinib mesylate (Gleevec, Glivec, formerly known as STI571), a relatively selective inhibitor of protein tyrosine kinases including platelet-derived growth factor receptor (PDGFR), would inhibit hCASMC proliferation and migration in vitro with little effect on endothelial cell proliferation and prevent restenosis in a swine balloon injury model. Methods. Proliferation and migration of stimulated human vascular smooth muscle and endothelial cells were quantified in cell culture in the presence of imatinib (0.001–10 µM). Imatinib-loaded drug-eluting stents were implanted in swine coronary arteries after predilatation with an oversized balloon, and neointimal proliferation was measured by quantitative angiography and histopathology. Results. Increasing doses of imatinib-inhibited autophosphorylation of the PDGFR and its downstream effects of proliferation and migration of human CASMC in a dose-responsive manner, yet had no effect on stimulated human aortic endothelial cells. However, imatinib-eluting stents had no effect on neointimal proliferation and restenosis in a standard porcine in-stent restenosis model compared to bare-metal or unloaded polymer-coated stents. Conclusion. We conclude that imatinib is a potent inhibitor of proliferation and migration of human vascular smooth muscle cells in vitro, but has no effect on human vascular endothelial cell proliferation. However, the lack of an in vivo effect on neointimal proliferation in a standard porcine coronary overstretch model does not support the use of imatinib mesylate for localized drug delivery in the prevention of in-stent restenosis.

       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

Figure 1

(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 cellular proliferation. Inset measures the IC50 of imatinib. (B) Imatinib does not inhibit human coronary artery endothelial cell (hCAEC) proliferation. Human CAEC were cultured for 48 hours in the presence of imatinib and 10% serum. All wells were normalized to the positive control. Error bars are SEM. *p < 0.05.

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)

Figure 2

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 highest dose. Imatinib had no effect on viability in hCAEC at any dose. Error bars are SEM. *p < 0.05.

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

Figure 3

(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 in the presence of increasing concentrations of imatinib. Inset measures the IC50 of imatinib. (B) Migration through an 8 µm pore Transwell filter was not affected by 10 µM imatinib. Human CAEC were placed on the top side of the filter and stimulated for 24 hours to migrate to the underside of the filter by 20 ng/ml VEGF and 1%BSA. Imatinib did not alter the migration through the filter. Error bars are SEM. *p < 0.05.

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

Figure 4

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.

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

Figure 5

Percent restenosis measured by quantitative coronary angiography 28 days after balloon injury and stent implantation. Error bars are SEM.

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

Figure 6

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.

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.

Results

       Inhibition of human vascular smooth muscle proliferation by imatinib. Human coronary artery smooth muscle cell proliferation was significantly increased compared to serum-starved cells and was reduced in a dose-dependent manner by imatinib with an IC50 of 0.058 µM. These results are based on 21 experiments (Figure 1A). Viability as measured by trypan blue exclusion test revealed no increase in cell death with increasing concentrations of imatinib except at the highest dose (Figure 2). Inhibition of human vascular smooth muscle migration by imatinib. Imatinib effectively inhibited human coronary artery smooth muscle migration as measured by cells moving from one side of an 8 µm pore Transwell filter to another. Without PDGF-BB stimulation, cell migration was not observed. Stimulation with 20 ng/ml PDGF-BB induced approximately 50% of the cells to move through the filter within 24 hours. Imatinib inhibited this migration in a dose dependent manner (Figure 3A). Imatinib has no effect on human coronary arterial endothelial cells. Imatinib had no effect on hCAEC proliferation when cells were grown in assay media (Figure 1B). Imatinib did not alter the viability of endothelial cells as measured by trypan blue staining (Figure 2). Imatinib also did not prevent VEGF-induced migration of hCAEC cells through an 8 µm pore Transwell filter (Figure 3B).
       Inhibition of PDGF-receptor tyrosine phosphorylation by imatinib. Stimulation of hCASMCs with PDFG-BB (50 ng/ml) and 10% FBS resulted in phosphorylation of the PDGF-b-receptor on tyrosine residues. The addition of imatinib to the cells prior to PDGF activation inhibited PDGF-b-receptor phosphorylation in a dose-dependent manner (Figure 4), with an IC50 of 0.028 µM.
       Polymer- and imatinib-coated stents did not prevent restenosis as measured by QCA. QCA was used to measure vessel diameter, balloon overstretch, stent implantation diameter and restenosis. The balloon was overstretched on average to 124 ± 3% of the artery size with no differences between treatments. Stents were implanted on average to 125 ± 4% of the native artery size in the bare-metal stent group, 118 ± 4% in the polymer-coated stent group and 109 ± 5% in the drug-coated stent group with a significant difference between the bare-metal and drug-coated stent groups (p = 0.035). The percent restenosis in the drug-coated stent group was 32 ± 6% compared to 20 ± 8% in the polymer-coated group and 9 ± 8% in the bare-metal group. While a trend towards increased restenosis was observed in the drug-eluting and polymer-coated stents relative to the bare-metal stent control arteries, there was no statistical difference between groups; p = 0.135 (Figure 5).
       Polymer and imatinib-coated stents did not prevent restenosis as measured by histomorphometric analysis. In-stent restenosis was 30 ± 11% in the bare-metal group, 16 ± 6% in the polymer-coated group and 25 ± 9% in the drug-coated group, which was not significantly different between treatments. The average area of the lumen was also not different between treatments (1.9 ± 0.7, bare; 1.3 ± 0.5, polymer; and 1.4 ± 0.5, drug in mm2). The average media and intima areas were not different between the groups, nor was the neointimal thickness (Figure 6).

Discussion

       The finding of late stent thrombosis following DES implantation will likely drive a continued search for a drug that potently inhibits VSMC proliferation without delaying stent endothelialization. This study demonstrates that low concentrations of imatinib inhibit the proliferation and migration of human coronary artery smooth muscle cells in tissue culture without significantly affecting endothelial cells. Imatinib meslyate’s wide therapeutic window between the IC50 of vascular smooth muscle and endothelial cell growth inhibition makes this DES strategy very appealing. However, in this study imatinib-coated stents did not inhibit neointimal proliferation in a swine balloon injury model.
       Myllarniemi et al¹⁸ previously demonstrated that orally administered imatinib (CGP 57148) inhibited neointimal formation in a rat aorta model of arterial injury, but did not quantify the IC50 of this response, nor the effect of imatinib on endothelial cell function. In contrast, oral imatinib failed to decrease restenosis after repeat treatment for in-stent restenosis,¹⁹ probably due in part to the inability to achieve similar serum levels of imatinib with tolerable systemic doses in humans. Our in vitro results are consistent with several other studies examining the effects of various inhibitors of the PDGF receptor (CGP 53716, PD 089828, CT52923 and AG-1295) on VSMC proliferation.^9–13,20 However, we demonstrate that imatinib is a significantly more potent inhibitor of the PDGF receptor and VSMC proliferation than previously studied tyrosine kinase inhibitors, with an IC50 more consistent with local tissue concentrations achievable by polymer-coated stent delivery.
       There are several possibilities for the lack of in vivo effect of imatinib on restenosis following DES implantation. Although we maximally loaded the polymer-coated stents with imatinib (600 µg/stent), it is possible that the dose was not high enough to achieve tissue concentrations sufficient to inhibit arterial smooth muscle cell proliferation in vivo. Both rapamycin and paclitaxel are significantly more potent inhibitors of VSMC in cell culture than imatinib, with IC50s of < 10 nM. Imatinib is also very water-soluble, perhaps resulting in poor tissue penetration and excessive drug washout into the circulation. In vivo tissue pharmacokinetics of imatinib and in vivo polymer-release characteristics were not examined in this study, and both factors could have substantial impact of the effectiveness of our drug on restenosis. It should be emphasized that the normal porcine artery model of restenosis is likely a limited assay of the effects of DES on restenosis in diseased human arteries. Finally, given the multiple cytokines and signal transduction pathways involved in VSMC proliferation following arterial injury,²¹ it is possible that selective inhibition of PDGF-R and c-KIT are insufficient to decrease neointimal formation.
       Given the clinical problem of late stent thrombosis following DES implantation, search for cell type-specific inhibitors that do not delay stent endothelialization should continue. Several analogs of imatinib have been developed to treat imatinib-resistant chronic myelogenous leukemia, and one analog (Dasatinib, BMS-354825) is a more potent inhibitor of PDGF-R than imatinib and has been shown to inhibit rat and human VSMC in vitro.²² The effects of this receptor tyrosine kinase inhibitor on endothelial cell function, in vivo neointimal proliferation, and in-stent restenosis have not been investigated.

Conclusion

       In summary, imatinib potently inhibited human CASMC proliferation and migration in vitro via inhibition of ligand-mediated PDGF-receptor autophosphorylation, while having almost no effect on endothelial cells at a 100-fold higher concentration. Unfortunately, imatinib-loaded DES did not inhibit in vivo neointimal proliferation in a standard porcine model of restenosis.