Effects Of Id1/NFκB/Survivin Signaling Pathway On EPCs Proliferation And Involved In The Repair Process After Vascular Injury

1. Background and ObjectiveAtherosclerosis is the pathological basis of many kinds of cardiovascular diseases suchas coronary heart disease (CHD),hypertension and diabetes, and which can lead to vascularstenosis eventually. Percutaneous coronary intervention (PCI) is a critical therapeutic optionin the treatment of vascular stenosis. However, PCI can not only bring benefits to patients,but also cause irreversible mechanical damage of the intimal, eventually resulting inendothelial damage and restenosis of the target vessel. Accelerated re-endothelialization canrestore the integrity of the structure and function of vascular endothelial, inhibitingneointimal hyperplasia and preventing restenosis. At first, endothelial repair mechanismsare considered to be mediated by neighboring endothelial cells (ECs), but ECs areterminally differentiated cells with limited capacity in endothelial repairing. More recently,increasing evidence suggests that endothelial progenitor cells (EPCs) have been found to bemainly endogenous repair mechanism after vascular injury. EPCs can home to sites ofinjury and differentiate into ECs and eventually participate in re-endothelialization aftervascular injury. However, the regulatory mechanisms of the proliferation and migration ofEPCs in re-endothelialization after vascular injury remain unclear. It is demonstrated thattumour-induced expression of inhibitor of DNA binding or differentiation1in EPCs andconditional Id1suppression impaired the proliferation of EPCs.Inhibitor of differentiation or DNA binding1(Id1) is an important subfamily memberof the helix-loop-helix (HLH) transcription factor family. Id1proteins are important forcell-cycle progression in several cell lines. Pertaining to recent studies, increased expression of Id1has been associated with the growth, proliferation, migration, andapoptosis of many kinds of cell lines. Recent studies have indicated that tumor can induceexpression of Id1in EPCs. And, it is documented that conditional Id1suppression canresult in impaired mobilization of EPCs. Moreover, over-expression of Id1has beendemonstrated to promote the survival and proliferation of spleen-derived EPCs in vitro. In aprevious study, we also demonstrated that Id1has an essential role in regulating theproliferation and migration of EPCs. However, the signaling mechanisms linked toId1-mediated EPC proliferation and vascular repair processes after injury have not beenaddressed.Recent studies have reported that Id1is possibly associated with thephosphatidylinositol-3-kinase (PI3K)/serine/threonine kinase Akt/nuclear factor kappa B(NFκB), PI3K/Akt, and NFκB/survivin signaling pathways in several cell lines. Severalstudies have indicated that PI3K is essential for Id1-mediated cell proliferation, migrationand survival. One of the important downstream signals of PI3K is Akt. Akt is transfered tothe membrane by direct binding to PI3K. It has been that activated Akt can phosphorylatemany kinds of proteins including Caspase9, eNOS, mTOR, GSK-3β, IκB, and NFκB. Inresting cells, NFκB is sequestered in the cytoplasm in an inactive state by bound to itsinhibitor IκB. Upon stimulation, IκB is degraded which frees NFκB and allows its nucleartranslocation and bind to target genes. Survivin is a recently described member of theinhibitors of apoptosis (IAP) family, which is an important regulator in the molecularmechanism of proliferation and apoptosis. Recent studies have reported that survivin isregulated by its interaction with many cell signaling pathways such as the mTOR, p21, andERK pathways. In addition, a recent study reported that survivin is under the control ofNFκB. However, the mechanism linked to Id1/NFκB/survivin pathway activation in EPCproliferation and vascular repair processes after injury are largely unknown.Therefore, the purpose of this study was to investigate whether there is anyrelationship between Id1/NFκB/survivin pathway activation and vascular repair processesafter injury.2. Methods2.1Isolation,culture and characterization of spleen-derived EPCs SD rat spleen-derived cells were isolated by density-gradient-centrifugation(Lymphoprep1.083, Tianjin, China) at1500rpm for20min. After purification with threewashing steps, the cells isolated were re-suspended in DMEM-L supplemented with10ng/ml VEGF and10%FCS. Then, incubate cells under an atmosphere of5%CO2at37℃.The medium with penicillin, and streptomycin was refreshed every3days. Forcharacterization, the cells cultured for about7days were fixed with4%Para formaldehyde,and then incubated with Dil-Ac-LDL and UEA-I, finally incubated with DAPI for5min.Triple-stained cells positive for UEA-I, Dil-Ac-LDL and DAPI were identified as EPCs.Additionally, FACS analysis was performed using antibodies CD34, CD133, VEGFR-2,and CD45.2.2Effect of Id1/NFκB/survivin on EPC proliferationTo investigate the effect of Id1/NFκB/survivin signaling pathway on SD ratspleen-derived EPCs, firstly, we transduced Ad-Id1and si-Id1into EPCs cultured for about4—7days, then we use the NFκB inhibitor BAY11-7082, the survivin inhibitor YM155toreduce the effects of Id1transfection. The EPCs treated above were trypsinised and thenplaced into96-well plates. Cell proliferation was measured by MTS assay.2.3Effect of Id1/NFκB/survivin on vascular repairTo determine the important role of Id1/NFκB/survivin signaling pathway inre-endothelialization after vascular injury in vivo, firstly, we examined the mRNA andproteins expression of Id1, NFκB, and survivin in injured carotid artery. Then, we injectedAd-Id1into balloon injured carotid, and employed the NFκB inhibitor BAY11-7082, thesurvivin inhibitor YM155to reduce the effects of Id1transfection. Evans Blue dye methodwas used to evaluate re-endothelialization after7,14,21days injury, and the neointimalformation was assessed at7,14,21days after vascular injury by staining with hematoxylinand eosin.2.4Western blot analysisProteins extracted from cells and injured vascular were measured by the Bradfordmethod. Equal amounts of the samples separated by a SDS-PAGE gel were subjected toelectrophoresis, and then transferred onto a PVDF membrane. The membranes wereblocked by2%BSA. The blots were incubated with primary antibodies including Id1, NFκB, and survivin. Finally, the detection of specific proteins was performed by enhancedchemiluminescence according to the manufacturer's instructions. The intensity of the bandswas quantified by Quantity One.2.5Reverse transcription-PCR (RT-PCR)Total RNA from EPCs and injured vascular was extracted following themanufacturer's instruction. RNA extracted above was reversely transcribed into cDNAusing a RT kit. The primer sequences for PCR were as follow:Id1: Upstream:5'-GGGCGAAGTGGTGCTTGGTCT-3'Downstream:5'-TGCGGTTCTGAGGCAGGGTAGG-3'NFκB: Upstream:5'-TCTGTTTCCCCTCATCTTTCC-3'Downstream:5'-GCGTCTTAGTGGTATCTGTGCTT-3'Survivin: Upstream:5'-GACCACCGGATCTACACCTTC-3'Downstream:5'-GAGTGCTTCCTATGCTCCTCTAT-3'GAPDH was used as control. cDNA was synthesized. The intensity of the bands wasquantified by Quantity One.3. Results3.1Isolation,culture and characterization of spleen-derived EPCsCultured for1day, the cells possessed a rounded shape. After5—7days of culture, thecells possessed cord-like, spindle-like or cluster-like structures. Fourteen days later, thereare colony-forming units and tubular-like appearance in cultured cells. For furthercharacterization, cells cultured for about7days were analyzed with immunofluorescenceand FACS. Cells, which stained positively for DAPI, Dil-Ac-LDL, and UEA-I andexpressed endothelial/stem cell markers including VEGFR-2, CD133, and CD34but notCD45were confirmed as EPCs.3.2Effect of Id1/NFκB/survivin on EPC proliferationTo determine the sub cellular localization of Id1, NFκB, and survivin in EPCs,immunocytochemistry was used. Our present results demonstrated that Id1, NFκB, andsurvivin were localized predominantly in the cytoplasm in the resting state.To investigate the effect of Id1/NFκB/survivin signaling pathway on EPC proliferation,firstly, Ad-Id1was transfected into EPCs. The results showed that there was increased NFκB and survivin proteins expression and increased nuclear translocation of NFκB. MTSassay was performed to examine the proliferation of EPCs. It showed that the proliferationof EPCs transfected with Id1was enhanced to about200%compared to control. Secondly,siRNA fragments were used to silence the Id1gene in EPCs. Si-Id1can cause about50%loss of Id1and survivin proteins expression and decreased nuclear translocation of NFκB.And, it can cause a decrease in EPC proliferation. Thirdly, the NFκB inhibitor BAY11-7082and the survivin inhibitor YM155were used to determine the role ofId1/NFκB/survivin signaling pathway in EPCs proliferation. The results showed that BAY11-7082and YM155can decreased the proliferation of EPCs. And, interestingly we foundthat there was an endogenous Id1and PI3K/Akt signaling pathway has an importantinteraction in EPCs.3.3Effect of Id1/NFκB/survivin on vascular repair3.3.1Expression of Id1, NFκB, and survivin in vascular repairIn this study we found that the expression level of Id1mRNA was very low inuninjured arteries. However, the expression of Id1mRNA was rapidly enhanced from6hafter vascular injury with a peak at24h and then gradually declined to the level of normal.The expression of Id1protein was upregulated from12h after vascular injury with a peak at14d and then gradually declined to the level of normal.The expression level of NFκB mRNA was very low in uninjured arteries. However, theexpression of NFκB mRNA was rapidly enhanced from6h after vascular injury with a peakat12h and then gradually declined to the level of normal. The expression of NFκB proteinwas upregulated from24h after vascular injury with a peak at7d and then graduallydeclined to the level of normal.The expression level of survivin mRNA was very low in uninjured arteries. However,the expression of survivin mRNA was rapidly enhanced from6h after vascular injury with apeak at2d and then gradually declined to the level of normal. The expression of survivinprotein was upregulated from24h after vascular injury with a peak at14d and thengradually declined to the level of normal.3.3.2Effect of Id1/NFκB/survivin on re-endothelializationEvans Blue dye method was used to determine re-endothelialization after7,14,21 days of injury. The white area at injured vessels was considered to be re-endothelialization,and the blue area at injured vessels was considered to be non-endothelialization. The resultsin this study showed that the re-endothelialized area in the Ad-Id1-injected group wasextensively larger than the control group. However, blockage of the NFκB/survivinsignaling pathway by BAY11-7082and YM155can abrogate Id1-inducedre-endothelialization after vascular injury in vivo.3.3.3Effect of Id1/NFκB/survivin on neointimal formationIn this study, the neointimal formation was assessed at7,14,21days after vascularinjury by HE assay. The result showed that there was a marked decrease in I/M ratio in theAd-Id1-injected group at14days compared with Ad-GFP-injected group. Blockage of theNFκB/survivin signaling pathway by BAY11-7082and YM155can abrogateAd-Id1-injected-induced decreased neointimal formation after vascular injury in vivo.4. Conclusions4.1Id1, NFκB, and survivin were present at EPCs and dynamically expressed inballoon-injured vascular;4.2Overexpression of Id1can promote EPCs proliferation, balloon-injured vascularre-endothelialization and inhibit neointimal formation at the early stage after vascularinjury via NFκB/survivin signaling pathway;4.3Blockage of the NFκB/survivin signaling pathway by BAY11-7082and YM155can abrogate Ad-Id1-induced EPCs proliferation, Ad-Id1-injected-induced balloon-injuredvascular re-endothelialization, and Ad-Id1-injected-induced decreased neointimal formationafter vascular injury;4.4Knockdown of Id1can decreased EPCs proliferation via NFκB/survivin signalingpathway.