| Clinical application of drug eluting stent (DES) has significantly reduced but notcompleptely sovled in-stent restenosis (ISR) for patients undergoing percutaneouscoronary intervention (PCI). Despite the benefits of DES, it also induces anotherunexpected issue, the in-stent thrombosis, which is associated with the delayedvascular healing or re-endotheliazation due to toxicities of the drugs and/or polymersused for manufacturing DES. The focal nature of restenosis makes it a highlyattractive target for gene therapy. Forecasting possibility of restenosis according togenes is conducive to enhancing values of risk stratification and providing morereasonable therapeutic options for patients. Gene therapies include transferringnucleotide, functional genes, or antisense oligonucleotides (ASODNs) and so on tosomatic cells of individuals to generate therapeutic effects. Many studies show thatASODNs has advantages over the traditional or other genic therapies due to thecombining with special target genes playing pivotal role in the disease process,thereby probably preserving more therapeutic efficacy and fewer side-effects. Hence,ASODNs as a candidate may be promising and particularily suitable for thedevelopment of next generation of DES.ISR is a complicated interaction process between the vessel wall and implantedstent (stent itself, polymers and drugs). The pathophysiological mechanisms of ISRmainly include damage of endothelial cells, aggregation of platelet and activation of inflammatory cells and subsequently over-expression of various growth factors.Despite other probable mechanisms, proliferation and transfer of vascular smoothmuscle cells (VSMCs) has been considered as the most important mechanisminvolved in ISR process. Accordingly, inhibiting the proliferation and transfer ofVSMCs is the key mechanism to prevent ISR. Growth transfer factor-β1(TGF-β1) isa member in the protein super-family, which plays an important role in regulating thedifferentiation, transfer, and apoptosis of VSMCs. Once vessel wall is injured,TGF-β1might serve as one of the dominant mediators stimulating proliferation andtransfer of VSMCs, leading to neoimtimal formation and thickening, vascular lumenloss and re-stenosis. Therefore, we hypothesized that ASODNs targeting TGF-β1could efficiently inhibit the expression of TGF-β1, thus reducing the proliferation andtransfer of VSMCs. The ASODNs targeting TGF-β1represents a promising feasibleagent to be used in drug eluting stent system for preventing ISR.The study consisted of two parts:(1) To investigate the effects of ASODNstargeting TGF-β1gene on inhibiting the proliferation and transfer of VSMCs in vitro;(2) To investigate the effects of ASODNs-drug eluting stent on preventing in-stentrestenosis and promoting reendothelialization in vivo. I. Effects of ASODNs targeting TGF-β1promoter gene on proliferation andtransfer of VSMCs in vitroObjectives: To synthesize ASODNs specifically silencing TGF-β1promoter gene andto observe its effectes on inhibiting the proliferation and transfer of VSMCs in vitro.Methods: HEK293cells were transfected with recombinant rabbit TGF-β1promoterplasmids to determine AP-1binding sites. ASODNs targeting rabbit TGF-β1was thensynthesized according to base pairing of nucleotide. VSMCs were cultured in vitroand identified by α-SMA immunoflurescence staining. After incubating with PDGF(10ng/ml), the in vitro study consisted of three groups: control group (only PDGF),PDGF+ASODNs (10ug/ml) group, PDGF+mismatch ASODNs (10ug/ml) group.TGF-β1expression was detected by west-blotting, cell proliferation and metabolismby5-BrdU infiltration method and WST-1method, cell transfer by Transwelltechnique.Results: Enzyme digestion of collagenase I was applied for the culture of normalVSMCs, which showed the typical pattern “Valley-peak”. α-SMA was abundant in theVSMCs by immunoflurescence staining.. PDGF at lower concentrations of10ng/mlrised the metabolic rate of VSMCs and promoted transfer and proliferation of VSMCs.ASODNs but not mismatch ASODNs significantly inhibted metabolic rate, transferand proliferation of VSMCs(P<0.01).Conclusions: ASODNs targeting AP-1enhancer locus of TGF-β1gene can efficiently silence TGF-β1gene and remarkably reduce the proliferation and transfer of VSMCs. II. Effects of ASODNs eluting stent targeting TGF-β1promotrer gene on in-stentrestenosis and reendothelialization in vivoObjectives: To produce ASODNs eluting stent targeting TGF-β1promotrer gene andto evaluate the potential effects of them on in-stent restenosis andre-endothelialization and to investigating the underlying mechanisms.Methods: ASODNs-drug eluting stent of2.0×12mm were developed with aconcentration of1.0μg/mm2ASODNs. Drug release dynamics of stent was measuredwith the use of HPLC in vitro. The ASODNs-drug eluting stent was then implantedinto aorta abdominalis of New Zealand rabbits. Rabbits were assigned to three groups:naked stent group (n=10), ASODNs-drug eluting stent group (n=10), and mismatchASODNs-drug eluting stent group (n=10). Three animals were sacrificed four weeksafter stent implantation; some tissue samples were preserved for HPLC detection, andsome for scanning electron microscope to observe stent surface endothelialization.Additionally, six animals were sacrificed eight weeks after stent implantation, and thestented segments of abdominal aorta were harvested with some samples preserved forHPLC detection and the renant samples for subsequent molecular and histologicalstudies. For molecular study, the samples were kept under the temperature of-80°C,mRNA and protein expression of TGF-β1and connective tissue growth factor (CTGF)were detected by real-time PCR and western blotting. For histological study, thesamples were embedded with methyl methacrylate, cutted into sections of5μm respectively from near, middle, and distant segment of stents, and then stained withHE and ETVG, the neointimal area and mean thickness was measured under themicroscope.Results: ASODNs eluting stent released more than90%of ASODNs within24h invitro. There remained some and trace ASODNs detected in local vascular tissuesrespectively after4and8weeks after stent implamtation. Scanning electronmicroscope showed re-endothelialization was nearly complete in all the three stentsgroups (n=3). Compared with the control group, mRNA and protein expressions ofTGF-β1and CTGF in naked stent group were upregulated obviously (P<0.01for all),ASODNs-drug eluting stent significantly reduced mRNA and protein expressions(P<0.01for all) of TGF-β1and CTGF. Compared with naked stent, there wassignificant reduction of neointima area (P<0.05) and restenosis degree (P<0.05for all)in ASODNs-drug eluting stent, and there were no differences of these indexesbetween the naked stent and mismatch ASODNs-drug eluting groups (P>0.05for all).Conclusions: Despite poorly controlled releasing of ASODNs with ASODNs-drugeluting stent in vitro, ASODNs-drug eluting stent targeting TGF-β1can inhibit mRNAand protein expression of TGF-β1, reduced in-stent restenosis without imparingre-endothelialization. |
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