Preparation And Biological Properties Of Sodium Ferulate Eluting Stent

Background and objective：Ischemic cerebrovascular disease is highly disabling and fatal disease, whichaccounts for70%-85%of cerebrovascular disease. About30%-60%of ischemic stroke isattributed to carotid artery stenosis. Surgical treatments of atherosclerotic arterial diseaseinclude balloon angioplasty and/or stenting, endarterectomy and open surgical bypass.However, restenosis following these interventions leads to reduced luminal diameter，ultimately occlusion and ischemia. The higher incidence of restenosis after surgery has aserious impact on the long-term benefits[1]. Stenosis is varying different degrees and quitecommon after cerebrovascular, cornary artery, renal artery and other peripheral vascularsurgery. Restenosis due to neointimal hyperplasia is known to occur after both balloonangioplasty and stent placement[2], neointimal hyperplasia is actually due to proliferationof smooth muscle cells in the media giving rise to appearance of fused intima and media,which is the universal response of a vessel to injury. Several factors contribute toneointimal formation. The endothelium is a critical factor protecting vessels fromthrombosis and atherosclerosis[4], endothelial monolayer was seriously impaired duringcatheterization and angioplasty[3], vascular endothelial cells(ECs) and smooth musclecells(VSMCs) are closely related, ECs might regulate vascular tone and proliferation ofVSMCs through nitric oxide, endothelin and prostacyclin pathway. Meanwhile VSMCsundergo a phenotypic transition into a synthetic phenotype, VSMCs acquire migratorycapacity in this transition; subsequently these cells migrate into the intimal area from tunica media, followed by SMC proliferation, are key events in early neointimal formation[3].Paclitaxel, sirolimus, zotarolimus and everolimus are used in durg eluting stent inorder to reduce in-stent restenosis. These drugs significantly inhibit proliferation ofVSMCs, but this uncontrolled, non-specific inhibition of cell proliferation impairs theprocess of re-endothelialization, and leads to lack of ECs growth, late restenosis and latethrombosis in previous studies[5-11]. An ideal and safe drug used in drug eluting stentshould inhibit neointimal hyperplasia and induce re-endothelialisation. Meanwhile,integrated and functional endothelium is importantly effective for preventing thrombosis[12-14]. Enhanced endothelialization may be an effective therapeutic alternative to reducerestenosis rate, also a key to clinical treatment.The sodium salt of ferulic acid, is one of phytochemical phenolic acids, has shownmultiple properties, including anti-tumor[15], antiatherogenic[13,14], anti-platelet effect[18,19], anti-oxidant[20]and anti-infammatory[21]. However, the possible roles and molecularmechanisms of SF to ECs have not yet been fully determined, we investigated the effect ofSF on HUVECs proliferation and miagration in vitro. The observation may providesexperimental and clinic basis for anti-restenosis. On the other hand, Sodiumferulate-eluting stent is made,which is consisted of a bare metal stent and polymer coatingmixed with sodium ferulate. Coating surface morphology, drug release kinetics, bloodcompatibility are evaluated in vitro.Methods：HUVECs were incubated with different concentrations of SF, and divided into6groups：0μg/ml，0.1μg/ml，1μg/ml，10μg/ml，100μg/ml and1000μg/ml. Cell viabilitywas evaluated by CCK-8kit at two time points (24and48hour). Cell migration distancewas evaluated by scratch wound healing assay．Expression of FoxM1and VEGF inHUVECs were detected through the Immunocytochemistry method, Western blot andimage analysis.On the other hand, sodium ferulate-eluting stents were made by dip coating process,coating quality and structural integrity were evaluated by microscope and scanning electron microscope. Drug release of sodium ferulate-eluting stent was calculated in an invitro system by high performance liquid chromatography; Hemolysis rate, prothrombintime and activated partial thromboplastin time were tested to confirm the bloodcompatibility of the self-made coating stent.Results：1. After24hours incubation with different concentrations of SF, proliferation ofHUVECs was significantly increased in10μg/ml and100μg/ml groups compare withcontrol group; After48hours, HUVECs viability trend is more obvious, cell viabilityincreased notably at the concentration of0.1-100μg/ml compared with0μg/ml. However,cell proliferation decreased significantly when SF was added at a concentration of1000μg/ml at24and48hours （P<0.05）.2. After12hours treatment with SF，cell migration distance was promoted in1-100μg/ml group compared with control group; After24hours treatment, cellularmigration distance was increased in0.1-100μg/ml group compared to0μg/ml; After48hours, cellular migration was still increased compared with control group, but migrationdistance was inhibited by SF at concentration of1000μg/ml after48hours treatment（P<0.05）.3.After12hours treatment, HUVECs with amplified expression of FoxM1in1-100μg/ml group had a brighter signal than control group. Also, the red fluorescence in0.1-100μg/ml was more brighter than control group （P<0.05）.4. The expression of FoxM1and VEGF was analyzed by Western blot. We found thatthe expression of FoxM1was markedly up-regulated in treated group(0.1-100μg/ml)compared with0μg/ml in12and24hours. The expression of VEGF also increased in10-100μg/ml group in12hours, this trend was more obvious in24hours, VEGF wassignificantly increased in0.1-100μg/ml group compared with0μg/ml （P<0.05）.5. PLGA50, PLGA75, PDLLA and PLLA were coated in a homogenous manner.Coatings are complete and uniform, few adhesion and bridging was found in the coating.No significant difference was found in surface morphology.6. PLGA50and PLGA75coating may have more sustained and stable drug release compare with PDLLA and PLLA group. More drug released from PLGA50and PLGA75group compared with PDLLA and PLLA group.7. Hemolysis ratio of each group (PLGA50，PLGA75，PDLLA and PLLA) was lessthan5%, which was considered safe in hemolysis. APTT and PT shows there were nosignificant difference between BMS and polymer coated stents.Conclusion：Sodium ferulate promotes HUVECs proliferation and migration in a does-dependentmanner, The up-regulation of FoxM1and VEGF are correlated with the proliferation andmigration of HUVECs. It suggests that SF may be an effective agent for restenosis.Surface morphology of4kinds of polymers coated stents show smooth and uniform, andhave good blood compatibility, while PLGA50and PLGA75stents are superior to PDLLAand PLLA in drug release kinetics.