A Novel PDGF-Receptor Blocker Eluted Stent Attenuates In-Stent Neointimal Formation In A Rabbit Carotid Model

Part 1:A novel PDGF-receptor blocker sunitinib inhibited proliferation and migration of vascular smooth muscle cells in vitro Aims:This study examined the effect of sunitinib on proliferation and migration of vascular smooth muscle cells(SMCs)as well as its underlying mechanisms.Methods:MTT assay was used to determine the cytotoxity of sunitinib on SMCs and endothelial cells(ECs).SMCs were stained with annexin V-FITC and propidium iodide(PI)for cell death detection.To examine the potential effect of sunitinib on the migration of SMCs,a scratch wound assay was first performed.a?actin of SMCs was stained in order to further characterize the mechanisms by which sunitinib inhibited SMC migration.Western blot was used to analysis PDGF-stimulated phosphorylation of extracellular signal-regulated kinases(p-ERK).Results:Sunitinib at concentration of 0.1,1 and 10?M significantly inhibited PDGF-induced SMC proliferation in a concentration-dependent manner through inducing necrosis,but,showed no adverse effect on ECs.In addition,sunitinib attenuated PDGF-stimulated SMC migration determined by scratch wound assay and a-action cytoskeleton staining.Moreover,we showed that sunitinib inhibited PDGF-induced phosphorylation of ERK in vitro.Conclusion:These results suggested that sunitinib inhibited SMC proliferation and migration through down-regulation of PDGF-induced p-ERK pathway.Thus this drug may be a promising candidate for drug eluting stent.Part 2:Fabrication of sunitinib eluting stent and evaluation of its biocompatibility in vitroAims:To fabricate sunitinib eluting stent and evaluate its drug loading capacity,biocompatibility and further characterize the influence on ECs seeded on the stent under the stimulation of fluid dynamics.Methods:Sunitinib dissolved in 500 ?l of ethanol was mixed with lubricating jelly to yield a drug concentration of 2 mg/gm of jelly and bare metal stent(BMS)was individually dipped in sunitinib jelly to produce a thin stent coating.The eluted amount of sunitinib was determined by measuring the additional weight of the coating.The hemocompatibility studied including platelet adhesion tests,RSMC attachment assay and assay examining proliferation of ECs on stents.Finally,sunitinib stents seeded with ECs were cultured under the stimulation of fluid dynamics,and mRNA and protein of ECs on the stent were analyzed later.Results:Incubation of the bare metal stents in the solution of sunitinib jelly resulted in coating of this compound in this preliminary experiment,and 32±5.4?g sunitinib was bound to the stent,and the amount of sunitinib on the stent before and after the expansion was unchanged.There were almost no adhered platelets on sunitinib eluting stent,but the platelets adhering on BMS showed spreading morphology.Level of SMC attachment on sunitinib eluting stent was lower than that on BMS(21.8±9.3 versus 11.2±5.4/strut,p=0.03).The mRNA and protein of ECs seeded on the stent were up-regulated in a time-dependent manner under the stimulation of fluid dynamics,which implied that sunitinib eluting stents can adapt to the environment of fluid dynamics.Conclusions:The newly fabricated sunitinib eluting stent showed excellent drug loading capacity,biocompatibility and can adapt to the environment of fluid dynamics.Part 3:Evaluation of sunitinib eluting stent in a rabbit carotid modelAims:We tested stent-based delivery of PDGF-receptor inhibitor sunitinib in the rabbit carotid model.Methods:Bare metal stent(BMS,n=12)and sunitinib coated stents(SES,n=12)were implanted in rabbit carotid model.Postoperatively,the implanted stents were monitored by a handle Doppler probe,and peak systolic velocity(PSV)was recorded every month.Diagnostic angiography was also performed to confirm the position and patency rate of the stents.1 month or 3 months after stent implantation,rabbits were euthanized and the stents along with the surrounding arteries were harvested.The arteries with stents were divided into three segments,and the first segment prepared for HE,the second for scanning electron microscopy(SEM),the third for western blot.Results:Twenty four stents were successfully implanted in the carotid artery of New Zealand rabbits as described in the experimental section,and both diagnostic angiography and Doppler ultrasound demonstrated the patency of the stent grafts.PSV didn't vary significantly between BMS and sunitinib eluting stent.After three months,there was obvious neointimal formation in artery treated with BMS,but the neointimal thickness was dramatically reduced in SES(BMS:0.22±0.05 versus SES:0.16±0.06 mm,P=0.03).The re-endothelialized areas for BMS and SES were as follows:BMS:91.7±3.7%,SES:89.5±6.8%(P=0.24).Phosphorylation of ERK at neointima site was significantly less in SES than in BMS,which suggested that sunitinib specifically inhibited PDGF-induced ERK activation.Conclusion:Sunitinib eluting stent attenuated neointimal hyperplasia,and unaffected re-endothelialization through suppression of PDGF-induced ERK pathway in SMC.This novel DES may become a potential candidate for treating vascular disorders.