TNF-? Promotes Survival And Migration Of MSCs Under Oxidative Stress Via NF-?B Pathway To Attenuate Intimal Hyperplasia In Vein Grafts

Background:In recent years,coronary heart disease(CHD)has become one of the main diseases endangering public health.Coronary artery bypass grafting(CABG)is the most effective method to treat serious CHD.Autologous vein grafts are commonly used for myocardial revascularization around the world.Disappointingly,even treated with many kinds of drugs,several lines of evidence suggest that approximately 20-40%of these vein grafts result in stenosis and even occlusion two years after the surgery.The therapeutic effect of CABG was severely influenced.Therefore,it is a very urgent problem to how to attenuate intimal hyperplasia in vein grafts.Intimal injury is usually considered as the start event for the development of intimal hyperplasia in vein grafts.Endothelial cells(ECs)serve as an important barrier,protecting against monocyte adhesion and the proliferation of vascular smooth muscle cells(VSMCs).The apoptosis of ECs will destroy the vascular barrier and expose VSMCs to the blood flow directly,leading to the adhesion and activation of inflammatory cells.Activated inflammatory cells then generate excessive reactive oxygen species(ROS),including H2O2 and superoxide anions(O2-).These ROS not only lead to cell apoptosis but also induce the production of inflammatory factors,which may gather more inflammatory cells at injury sites.This vicious cycle results in the production of massive ROS,which can constantly aggravate oxidative damage to ECs and delay the re-endothelization,and finally lead to intimal hyperplasia.Mesenchymal stem cells(MSCs)can be recruited from bone marrow to peripheral blood circulation.Then MSCs can home to injured intima,differentiate into mature endothelial cells,and promote endothelial repair.The migration of MSCs to injured intima is the important prerequisite that MSCs play the role of promoting tissue repair.It is widely acknowledged that SDF-la/CXCR4 axis plays a critical role in regulating the migration of stem cells.Therefore,upregulating the expression of CXCR4 in MSCs could promote MSCs to migrate to injured intima.However,some studies find that oxidative stress exists in vascular intima and the blood circulation following both vein grafting and vascular injury,after a short period of time and lasting for several weeks.In rat models of acute balloon injury of common carotid artery,we also find that oxidative stress is involved in the intimal hyperplasia.Under oxidative stress environment,large quantities of ROS,including H2O2 and superoxide anions,were generated.The excessive and/or persistent stimulation of oxidative stress can reduce the function of MSCs,and increase apoptosis.As a consequence,the therapeutic effect of the transplantation of MSCs can be seriously reduced.The NF-?B(nuclear factor of kappa B)pathway widely participates in gene expression and regulation of chemokines,cytokines and adhesion molecules.The I?B kinase(IKK)complex consists of two catalytic subunits(IKK-a and IKK-?)and a regulatory subunit(IKK-y/NEMO).IKK-a and IKK-? are critical for NF-?B to perform its regulatory function.Bcl-2(B-cell lymphoma-2)gene could encode the Bcl-2 protein,which has been thought of as an anti-apoptotic protein.And the Bcl-2 promoter region has been shown to contain NF-?B response elements.In theory,NF-?B could attenuate cell apoptosis by up-regulating the expresson of Bcl-2 and other anti-apoptosis genes.Other studies have found that the NF-?B pathway is related to regulation of directional migration.Therefore,we attempt to activate the NF-kB pathway by stimulation with TNF-a,a kind of important proinflammatory factor,to promote survival and migration of MSCs under oxidative stress and attenuate intimal hyperplasia in vein grafts.The purposes of the study are to observe the expression of total CXCR4 and the CXCR4 on the surface of MSCs,migration,proliferation and secretive function of MSCs,via TNF-a preconditioning(PC)MSCs.We can determine the regulation of TNF-a to MSCs,and observe the role of NF-?B pathway played in the changing process of several biological functions.Then,we could further explore the role of miRNA played in TNF-a regulating NF-?B pathway via screening for miRNA which expression is changed significantly in these miRNAs targeted with IKK-a.We wish to determine the role of TNF-a promoting survival and migration of MSCs under oxidative stress via NF-?B pathway,and observe the effect of MSCs preconditioned with TNF-a on attenuating intimal hyperplasia in vein grafts.In general,this research mainly aims to explore the effect and mechanism of TNF-a promoting survival and migration of MSCs under oxidative stress,and determine whether TNF-a preconditioning could improve the therapeutic effect of MSCs on intimal hyperplasia in vein grafts.The content of this study is mainly divided into three parts as follows.Part one:TNF-? promotes survival and miration of MSCs under oxidative stress via NF-?B pathwayObjective:To investigate whether tumor necrosis factor ?(TNF-?)can promote the migration,proliferation and secretion of MSCs,and explore further the effect of TNF-?on survival and migration of MSCs under oxidative stress,and establish what the role of NF-?B pathway plays in these regulation processes at the same time.Methods:1.We preconditioned(PC)MSCs with TNF-a(TNF-?-PCMSCs)for 24h with different concentrations(0,10,30,50ng/ml)and measured the expression of NF-?B-p65 and p-NF-KB-p65 to choose the optimal TNF-? concentration.2.Treatment with IKK ?(NF-?B inhibitor,5?M)was carried out prior to TNF-a treatment for 30min,western blot was used to measure the expression of p-IKK-?/IKK-? and p-IKK-?/IKK-? to determine the activation of TNF-? to NF-?B pathway.Experimental grops were divided as follows:?.Control group,treated with?-MEM+10%FBS,without IKK ? and TNF-?;?.TNF-? group,MSCs were treated with TNF-?(50ng/ml)for 24h;?.IKK ?+TNF-? group,treated with IKK ?(NF-?B inhibitor,5?M)prior to TNF-a treatment for 30min to antagonize the NF-?B pathway,then MSCs were treated with TNF-a(50ng/ml)for 24h;?.IKK ? group,MSCs were treated with IKK ?(5?M)for 24h.3.Flow cytometry was used to detect the effect of TNF-? on cell cycle of MSCs;CCK-8 was applied to detect the change of total cell activity;EDU assay was used to observe the proliferation of MSCs.4.Treatment with IKK ?(5?M)and SB203580(p38 MAPK inhibitor,5?M),western blot was used to observe the expression of p-p38 and p38 to determine the role of TNF-a to p38-MAPK pathway;CCK-8 assay detects the effect of NF-?B pathway and p38-MAPK pathway on cell activity.5.Western blot detects the expression of p-NF-?B-p65/NF-?B-p65 and CXCR4.Cell-surface phenotype analysis also was used to detect the expression of CXCR4 on the surface ofMSCs to determine the regulation role of NF-?B pathway to CXCR4 expression.Transwell assay was applied to evaluate the effect of TNF-a on migration of MSCs.6.Annexin V-FITC/PI flow cytometry detects the apotosis of MSCs under oxidative stress in different groups.Western blot observes the expression of Bcl-2 and CXCR4 of MSCs under oxidative stress in different groups to explore the impact of oxidative stress on MSCs,and the role of TNF-a treatment to survival and migration of MSCs.Results:1.The expression of p-NF-?B-p65 increased gradually with the concentration of TNF-a was adding.The effect was dose-dependent,and 50ng/ml TNF-a could significantly activated the NF-?B pathway.2.IKK-a and IKK-? are pivotal upstream regulatory proteins of the NF-?B pathway.TNF-a increased phosphorylation of IKK-a and IKK-?,with activation was effectively antagonized by IKK XII.3.Flow cytometry suggested that the percentage of cells in S phase was increased by TNF-a activating NF-?B pathway,which could reflect that cell proliferation was enhanced in some extent.CCK-8 assay suggested that TNF-a increased total cell viability,which was reduced by IKK ? antagonizing the NF-?B pathway.Total cell viability of IKK?+TNF-? group was reduced compared with IKK ? group.The EdU assay results suggested that TNF-a promoted cell proliferation,which was reduced by IKK ?antagonizing the NF-?B pathway.4.TNF-? also could activate the p38-MAPK pathway,and the activation can be antagonized by SB203580.Following IKK XII antagonizing TNF-a activated NF-?B pathway,cell viability was raised by SB203580 blocking P38-MAPK pathway.5.TNF-a increased p-NF-?B-p65 and CXCR4 expression The expression levels of p-NF-KB-p65 and CXCR4 had almost the same change trend.Transwell assay suggested that TNF-? improves migration of MSCs towards SDF-la.IKK XII could reduce the improvement of migration caused by TNF-?.6.Flow cytometry demonstrated that oxidative stress gave rise to significant apoptosis of MSCs.Preconditioning with TNF-? significantly reduced apoptosis caused by oxidative stress.Following treatment with oxidative stress,the expression levels of Bcl-2 and CXCR4 were markedly decreased.Preconditioning with TNF-a resulted in the ove rexpress ion of Bcl-2 and CXCR4 under oxidative stress,and these effects can be abolished by IKK XII.Conclusions:TNF-a activates NF-?B pathway by increasing phosphorylation of IKK-a/p.Preconditioning with TNF-a can promote survival and migration of MSCs.The NF-?B pathway plays the critical role in regulating these processes.TNF-a can reduce apoptosis and increase migration of MSCs under H2O2-induced oxidative stress via the NF-?B pathway.Part two:miR-23b-3p participates in the regulation of NF-?B pathway activated by TNF-?Objective:To investigate the role of microRNA(miRNA)played in the regulation of NF-?B pathway activated by TNF-a in MSCs.To screen for miRNAwhose expression is changed significantly in these miRNAs targeted with IKK-a,and explore the relationship between miRNA and NF-?B pathway as well as the downstream fimctional activity via endogenous and exogenous tests.Methods:1.Choose out the miRNAs which take IKK-a as target gene,according to the prediction that between miRNA target gene and binding sites on several websites,such as TargetScan 7.1,miRbase and microRNA.org.Then we choose these miRNAs as study object,and qRT-PCR was used to detect the expression change of these miRNAs after TNF-a treatment to screen for the most related miRNA.2.Synthesise the mimic and inhibitor of target miRNA,and synthesise the wild-type and mutant-type double luciferase reporter plasmid of IKK-a.Dual luciferase reporter assay was used to verify whether the IKK-? was the target gene of the miRNA we studied.3.Western blot was applied to determine the relationship between the miRNA and NF-?B pathway as well as the expression of CXCR4.4.Transwell assay tests the effect of transient transfection miRNA inhibitor to the migration of MSCs.Results:1.According to website prediction and qRT-PCR analysis,we find that the expression of miR-23b-3p is changed significantly in these miRNAs targeted with IKK-? after TNF-? treatment.Therefore,miR-23b-3p will be taken as the study target.2.miR-23b-3p could significantly inhibit the expression of firefly luciferase in the cells transfected with wild-type reporter plasmid,but not in the cells transfected with mutant-type reporter plasmid.Dual luciferase report system exogenously verified that IKK-a was the target gene of miR-23b-3p.3.miR-23b-3p mimic could down-regulate the activity of NF-?B pathway and the expression of CXCR4.miR-23b-3p inhibitor could up-regulate the activity of NF-?B pathway and the expression of CXCR4.4.Transwell assay verified that transient transfection with miR-23b-3p inhibitor could promote the migration of MSCs.Conclusions:TNF-a could down-regulate the expression of miR-23b-3p and attenuate the inhibition effect of miR-23b-3p to IKK-a,thereby activate NF-?B pathway and promote the migration of MSCs.miR-23b-3p participates in the regulation of NF-?B pathway activated by TNF-?.Part three:The effect of MSCs preconditioned by TNF-? in treating intimal hyperplasia in vein graftsObjective:To investigate whether preconditioning with TNF-a can promote the survival and migraton of MSCs in experimental animal models.To explore the effect of MSCs preconditioned by TNF-a in treating intimal hyperplasia in vein grafts.Methods:We preconditioned(PC)MSCs with TNF-a(TNF-?-PCMSCs)for 24h and labeled MSCs with CM-Dil.Then CM-Dil labeled TNF-?-PCMSCs were collected and injected into the vein graft rat models through the caudal vein.2.Rat blood samples were obtained 1 day and 1 week after vein grafting,and serum isolated from each tube after centrifugation.Methane dicarboxylic aldehyde(MDA)concentrations in the serum were measured to assess the level of oxidative stress using an ELISA kit.3.MSCs were preconditioned for 24h and the culture medium of each well collected.VEGF and HGF protein concentrations in each well were quantified using an ELISA kit.4.Part of the rats was humanely killed 1 week after operation,the specimens were made into frozen sections and MSCs homing was observed using fluorescence microscopy.5.TUNEL assay was put into use to evaluate the endothelial apoptosis of vein graft.6.All remaining rats were sacrificed 4 weeks after the operation.The specimens were made into paraffin sections.Van Gieson staining was performed in order to evaluate intimal hyperplasia.Results:1.Living cell tracer CM-Dil could label MSCs successfully,and it does not affect cellular activity.2.An ELISA assay indicated that the serum level of MDA was significantly increased 1 day after vein grafting,giving positive confirmation that oxidative stress resulted from vein grafting in the rat blood circulation.3.MDA concentration was significantly reduced by treatment of TNF-?-PCMSCs 1week after vein grafting compared with Non-PCMSCs group.4.The TUNEL assay revealed that endothelial cells become apoptotic after vein grafting,but TNF-?-PCMSCs treatment could significantly delay the apoptosis of endothelial cells.5.The secretions of VEGF and HGF were both significantly elevated in TNF-?-PCMSCs with or without H2O2 induced oxidative stress.6.Homing of MSCs was observed in the vein grafting models.Migration of TNF-?-PCMSCs to the intima of the vein graft was significantly increased compared with Non-PCMSCs,an effect that was reversed by pretreatment with IKK XII.7.Van Gieson staining demonstrated that the neointima of the TBNF-?-PCMSCs group was also thinner than that of the Non"PCMSCs group,intimal hyperplasia in the vein graft was significantly reduced.Conclusions:There was oxidative stress resulted from vein grafting in the rat blood circulation.Precondiitoning with TNF-? can promote survival and migration of MSCs under oxidative stress,then lead to the more number of TNF-?-PCMSCs migrating to the intima of the vein graft,and promote endothelialization of blood vessels.Pretreatment with IKK XII could reverse these effects,confirming that the NF-?B pathway plays a critical role in regulating these processes.Moreover,TNF-?-PCMSCs Ccould reduce the apoptosis of endothelial cells via secretions of VEGF and HGF.The reduction of apoptosis of endothelial cells could lower oxidative stress level.The two together promote the melioration of intimal hyperplasia in vein grafts.TNF-a preconditioned MSCs could more efficiently attenuate intimal hyperplasia in vein grafts.Innovation and significanceThis study confirms that TNF-a activates NF-?B pathway by increasing phosphorylation of IKK-?/?.Preconditioning with TNF-a can promote survival and migration of MSCs.The NF-?B pathway plays the critical role in regulating these processes.miR-23b-3p participates in the regulation of NF-?B pathway activated by TNF-?.The complex mechanism of miRNA regulating pathway need to be explored further.Preconditioning with TNF-? can promote survival and migration of MSCs under oxidative stress,then lead to the more number of TNF-?-PCMSCs migrating to the intima of vein grafts.TNF-a preconditioned MSCs could more efficiently attenuate intimal hyperplasia in vein grafts,and improve the effect of MSCs preventing vascular restenosis.This study provides theoretical basis and new ideas for improving the therapeutic effect of MSCs transplantation.