| Background: The autologous saphenous vein remains a widely used conduit for coronary artery bypass grafting(CABG).Occlusion of venous grafts is observed in approximately15% to 25% of patients during the first postoperative year and over 50% after ten years.Today,vein graft failure occurs as a result of neointimal hyperplasia remains an important and unresolved problem of cardiovascular surgery.The main pathogenesis of vein graft restenosis is neointimal hyperplasia associated with vascular smooth muscle cell(VSMC)migration and proliferation.Phenotypic Switched VSMC migration from the media into the intima and proliferation in response to the release of a variety of mediators,growth factors,and cytokines is the key event in the development of neointimal hyperplasia.Increasing evidence indicates that miRNA regulate key genetic programs in cardiovascular biology,physiology,and disease.miRNA also play important roles in the control of VSMC phenotypic switch,migration and proliferation.miR-17~92 cluster is one of the miRNA cluster and has been identified as an important regulator for cell growth,differentiation,and apoptosis.miR-221 also have effects of pro-proliferation,pro-migration,and anti-apoptosis in VSMC via the target gene of p27(Kip1)and p57(Kip2).Objective: We hypothesis that dysregulation of miR-17~92 cluster function may lead to VSMC phenotypic switch and the vascular neointimal hyperplasia formation.Therefore,the objective of the study was to evaluate the role,if any,of miR-17~92 cluster in VSMC phenotypic switch in vitro and in response to neointimal hyperplasia lesion formation after vein graft in vivo.To further confirm the effect of miRNA Sponge on inhibition of neointimal hyperplasia formation,we applied miR-221 Sponge to reduced the neointima formation in vivo.Methods: Rat model of vein graft restenosis established interposition bypass grafting from the autologous jugular vein to the carotid artery.Total RNA was extracted with Trizol reagent.RNAs obtained from vein grafts at postoperative 7 days,14 days and 21 days were subjected to the MicroRNA Microarray analysis.qRT-PCR for miRNA was performed using Hairpin-it miRNAs RT-PCR Quantitation Kit and SYBR Green Master Mix Kit.Primary rat VSMCs were isolated from aortas of male SD rats.We constructed an adenovirus vector expressing miR-17~92 using AdMax system.To investigate the potential link between VSMC proliferation and miR-17~92 expression,the expression of pre-miR-17~92 was determined in nonproliferative VSMCs and in proliferative VSMCs stimulated by either platelet-derived growth factor(PDGF)-BB(20 ng/mL)for 4h,6h,8h,12 h,24h,48 h and 72 h or differetnt dose PDGF-BB1(0 ng/ml,20ng/ml,50 ng/ml and 100 ng/ml)for 24 h.To further examine the functional role of miR-17~92 in VSMCs,we sought to determine whether inhibition of miR-17~92 could affect PDGF-BB induced VSMC proliferation.To this end,we applied miR-17~92 and miR-221 Sponge technology to knock down the expressiong of miR-17-92.In subsequent experiments,we determined the effect of on VSMC proliferation by using 2 different methods: cell counting and BrdUrd incorporation assay.Cell migration was performed by the SMC scratch wound assay and transwell chambers.To identify the potential molecular targets of miR-17~92 that may contribute to miR-17~92 cluster mediated cellular effects,bioinformatics databases such as TargetScan,miRanda,miRDB,PicTar,miRGen were used.To verify the potential target genes of miR-17~92,both gain-of-function and loss-of-function approaches were applied.To determine the effect of the miR-17~92 and miR-221 Sponge on neointima formation and vascular remodeling,the vein grafts were isolated after treatment for morphometric analysis,immunohistochemistry was used to determine the proliferation and immunofluorescence to determine the phenotypic switch in vivo.Finally,We applied miR-221 Sponge to reduced the neointima formation in vivo,which to further confirm the effect of miRNA Sponge on inhibition of neointimal hyperplasia formation.Results: Compared with normal jugular vein,microarray analysis demonstrated that aberrant miRNA expression was a remarkable characteristic in vein graft walls after bypass grafting.miR-17~92 was found to be significantly up-regulated in vein grafts was verified by qRT-PCR.The expression of pre-miR-17~92 in PDGF-BB treated VSMCs was significantly higher than that in vehicle-treated VSMCs,which was time-dependent.The dose-dependent response to PDGF was measured in cultured VSMCs after 24 hours of treatment with different concentrations of PDGF.Gain-and loss-of-function studies shown that knockdown of miR-17~92 cluster expression significantly inhibits migration and proliferation of cultured VSMC.To explore the relationship between miR-145 and the VSMC phenotype,we applied a well-established VSMC model for phenotypic modulation in which VSMC dedifferentiation was induced by PDGF-BB.VSMC differentiation marker genes such as SMA,calponin,and SM-MHC were downregulated both at the mRNA and protein levels by PDGF.Knockdown the miR-17~92 by miRNA Sponge significantly increased the VSMC differentiation marker genes compared with those from vehicle or control adenovirus-treated PDGF.Furthermore,we primary identified the PTEN as a potential target gene of miR-17~92 cluster.Finally,knockdown of miR-17~92 via miRNA Sponge in rat vein grafts suppressed VSMC proliferation in vivo and neointimal lesion formation after bypass grafting.miR-17~92 sponge treatment was also improved vein grafts haemodynamics and increased the VSMC differentiation marker genes in vivo.Finally,miR-221 sponge therapy can significantly reduce miR-221 activity and inhibit neointimal hyperplasia in vein graftsConclusion: The present study show that miR-17~92 cluster is a key regulator of vascular smooth muscle proliferation and migration in vitro,and neointimal lesion formation in vivo.We also found that knockdown the expression of miR-17~92 markedly increased the expression of VSMC differentiation markers,such as SM22 a,SM a-actin,and calponin markers,suggesting that miR-17~92 cluster may also play a critical role in VSMC phenotypic switch.These results suggest the potential for miR-17~92 and miR-221 sponges promising gene therapy for preventing vein graft failure. |
