Effects Of Schlafen5 On Proliferation And Migration Of Endothelial Progenitor Cells And Its Role And Mechanism In Vascular Injury Repair

1 Background and ObjectiveCoronary atherosclerotic heart disease(CHD)is mainly caused by atherosclerosis,and endothelial damage is a risk factor for atherosclerosis.Current treatments include risk factor control and percutaneous coronary intervention(PCI).However,lumen restenosis may occur in some patients after PCI,so novel treatment strategies are needed.Endothelial progenitor cells(EPCs),which originate from spleen and bone marrow,can homing to the damaged blood vessels and differentiate into endothelial cells,suggesting that EPCs play a role in vascular repair.However,the basic mechanism involved in EPCs attribute regulation remains uncertain.Schlafens(Slfns)(sleep factors)are a class of highly conserved proteins that control the development and maturation of T cells and impair T cell proliferation and activation when overexpressed.Slfn5 has been reported to regulate the expression of matrix metalloproteinases(MMP)genes involved in RCC migration,such as MMP-1and MMP-13.Thus,Slfn5 is a protein with extremely important functions,which is of great significance for its further study.However,our previous study found that Slfn5 was expressed in rat EPCs,and it is still unclear whether Slfn5 is involved in the regulation of EPCs.Therefore,we proposed the hypothesis that Slfn5 may be involved in the regulation of EPCs proliferation,migration and other biological patterns,thus affecting its ability to repair injured blood vessels.2 methods2.1 Bioinformatics analysis of Slfn gene family in vertebratesThe gene structure,chromosomal localization,tissue specificity of expression,and physicochemical properties of the encoding proteins of Slfn family genes in human and mouse were analyzed by bioinformatics.Then,the phylogenetic tree of Slfn gene family members from 27 species was constructed,and the gene structure and conserved motif were analyzed.2.2 Construction of sh RNA-Slfn5 recombinant Adenovirus vectorFive si RNA fragments were designed and synthesized according to the m RNA sequence of RAT Slfn5 gene,which were cloned into PDKD-CMV-U6-sh RNA and transferred into competent E.coli cells.The transformants were screened by PCR,and the positive clones were sequenced.The target shuttle plasmid sh RNA-Slfn5(5)was selected by ADMAX system to package and amplify the recombinant adenovirus sh RNA-Slfn5 and the virus titer was determined.The target gene of Slfn5 overexpressed adenovirus plasmid was used to carry Flag tags,and Flag expression in293 T cells was detected by Western blot to observe the effect of target plasmid on Slfn5 expression.Finally,EPCs were infected with virus and the transfection efficiency was measured by the amount of green fluorescent protein.2.3 Effect of Slfn5 on EPCs proliferation and migration and its mechanism2.3.1 Influence of Slfn5 on EPCs proliferation and migration2.3.1.1 EPCs culture and identificationThe spleen-derived monocytes were isolated by density gr Adient centrifugation.The cells positive for Ac-LDL-DI and FITC-UEA-I staining were identified as differentiated endothelial progenitor cells.In Addition,the expression of endothelial markers(CD31,KDR)and progenitor markers(CD34,CD133,CD45)were assessed by flow cytometry.The expression of VWF,CD34 and CD133 was identified by immunofluorescence.2.3.1.2 Determination of transfection efficiency of EPCs transfected with sh RNA-Slfn5 and Ad-Slfn5The expression of green fluorescent protein was observed under a fluorescence microscope,the percentage of green fluorescent cells in all cells was calculated,and the cell transfection efficiency was calculated.Slfn5 m RNA expression level was detected by RT-PCR.2.3.1.3 The effect of Slfn5 on EPCs proliferation and migration was observed at the cell level in vitroEPCs derived from rat bone marrow were isolated and cultured for 6-8 days for the experiment.The experimental groups were as follows:(1)Ad-Slfn5(Slfn5overexpression group);Sh RNA-Slfn5(Slfn5 interference group);(3)Ad-control(overexpression control group);(4)sh RNA-control(interference control group);(5)Control(untreated EPCs blank control group)sh RNA-Slfn5,Ad-Slfn5 and corresponding empty virus control adenovirus were transfected into cells and used for experiment 48 hours after transfection.The proliferation of EPCs was detected by CCK8 method,the cell cycle distribution was detected by flow cytometry,and the migration ability of cells was detected by Transwell chamber.2.3.2 The mechanism of Slfn5 regulating EPCs was observed at the cell level in vitro EPCs were transfected with sh RNA-Slfn5,Ad-Slfn5 and the corresponding air virus control adenovirus.Forty-eight hours after transfection,the protein level and m RNA level of MT1-MMP in EPCs were detected by Western blot and RT-PCR.2.3.3 The effect of Slfn5 on the repair of carotid artery injury was observed at the level of animal modelMale SD rats were anaesthetized with 75mg/ kg pentobarbital and heparinized with 100 UI/ kg heparin sodium.Balloon injuries were performed three times in the left common carotid artery with a 2Forgarty catheter.Immediately after balloon injury,pre-transfected Ad-Slfn5,sh RNA-Slfn5,Ad-control,sh RNA-control EPCs and normal EPCs were injected through the tail vein.The control group was injected with normal saline.Postoperative penicillin was used to prevent infection.Evin blue staining was used to observe the re-endothelialization at 14 days after injury,and HE staining was used to observe the proliferation of new intima at 14 days after injury.3 Results3.1 Bioinformatics analysis of Slfn gene family in vertebratesThere were significant differences in gene structure and tissue distribution of expression among members of human Slfn family.Its coding protein is mostly acidic,high hydrophilic.Phylogenetic analysis showed that Slfn members were clustered in four major lineages,three of which had strong guidance support(90% to100%).Tissue distribution of human Slfn family expression indicated that Slfn 5 was mainly expressed in transformed skin fibroblasts,subcutaneous adipose tissue,lung and spleen.Gene structure and conserved motif analysis showed that the motifs of Slfn members were roughly the same.3.2 Construction of rat sh RNA-Slfn5 recombinant Adenovirus vectorSequencing confirmed that 5 groups of target plasmids were successfully constructed.Recombinant adenovirus plasmids sh RNA-Slfn 5(1),sh RNA-Slfn5(4)and sh RNA-Slfn5(5)could significantly inhibit the expression of Flg protein.The titer of sh RNA-Slfn5 virus was 3.95×1010 pfu/m L.The transfection efficiency of EPCs was(85.64±2.58)%.3.3 Influence and mechanism of Slfn5 on EPCs proliferation and migration3.3.1 Influence of Slfn5 on EPCs proliferation and migration3.3.1.1 EPCs culture and identificationBone marrow-derived EPCs of rats were successfully isolated and cultured.They were fusiform,oval and polygonal,and typically showed clonal,line-like and vascular annular growth.UEA-I and Dil-Ac-LDL were stained and identified.Observation under confocal laser showed that the cells ingesting Dil-Ac-LDL showed red fluorescence,the cells ingesting UEA-I showed green fluorescence,and the cells ingesting Dil-Ac-LDL and UEA-I showed yellow fluorescence,that is,the cells were double-stained positive.Denotes differentiating EPCs(N > 90%).The percentages of CD31,CD34,CD45,KDR and CD133 positive cells were(99.70±3.84)%,(83.90±2.65)%,(0.80±2.82)%,(97.40±2.76)%,(81.60±2.84)%,respectively.VWF(88.29±2.31)%,CD34(84.90±2.15)%,CD133(83.70±2.15)% were identified as endothelial progenitor cells by immunofluorescence.3.3.1.2 Determination of EPCs transfection efficiency by sh RNA-Slfn5 and Ad-Slfn5 transfectionUsing EPCs cultured for about 8 days,sh RNA-Slfn5 virus and Ad-Slfn5 virus were transfected:Slfn5 expression in the transfected sh RNA-Slfn5 group was significantly down-regulated compared with that in the control group(sh RNA-Slfn5 vs sh RNA-control 0.598±3.564 vs 0.998±1.231)(P < 0.05).The expression of Slfn5 in the transfected group with Ad-Slfn5 virus was significantly upregulated compared with that in the control group(Ad-Slfn5 vs Ad-control 23.234±2.241 vs 1.078±1.983),which proved that the virus was successfully transfected by EPCs.3.3.1.3 Effect of Slfn5 on EPCs proliferation and migrationEPCs were transfected with sh RNA-Slfn5,sh RNA-control,Ad-Slfn5 and Ad-control,and cell proliferation,migration and cell cycle analysis were detected 48 h after transfection.The proliferation of Slfn5 knockdown group was significantly increased by CCK8 assay(sh RNA-Slfn5 group vs sh RNA-control group,1.63±0.05 vs 0.98±0.07,P < 0.01)(n=6).The cell proliferation ability of the Slfn5 overexpression group was significantly decreased(Ad-Slfn5 group vs.Ad-control group,1.53±0.09 vs.1.67±0.22,P < 0.01)(n=6).Transwell migration test results show that:The migration of EPCs transfected with Ad-Slfn5 virus was significantly lower than that of Ad-Control group(Ad-Slfn5 group vs Ad-Control group,28.17±3.56 vs58.17±3.28,P < 0.01)(n=6).The migration ability of EPCs transfected with sh RNA-Slfn5 virus was significantly higher than that of the sh RNA-control group(sh RNA-Slfn5 group vs sh RNA-control group,94.33±1.89 vs 55.50±2.87,P < 0.05)(n=12).Cell cycle analysis by flow cytometry showed that,compared with the Ad-control group,the over-expression of Ad-Slfn5 increased the proportion of G1 stagnated EPCs,but reduced the proportion of G2+S stagnated EPCs.Compared with sh RNA-control group,sh RNA-Slfn5 significantly increased the proportion of cells in the G2+S phase.3.3.2 The mechanism of Slfn5 regulating EPCsAfter about 8 days of EPCs culture,after 48 hours of different treatment:(1)control group;(2)Ad-Slfn5 transfection group;(3)Ad-control transfection group;(4)sh RNA-Slfn5 transfection group;(5)In sh RNA-control transfection group,the levels of MT1-MMP were analyzed by WStern blot and RT-PCR.RT-PCR and WB results showed that compared with the control group,Ad-Slfn5 transfection significantly reduced the expression of MT1-MMP protein and m RNA(m RNA level: 0.3388±0.02 vs.1.113±0.05,n = 3,P <0.05;Protein level: 0.45±0.03 vs.0.8421 ±0.06,n = 3,P<0.05);In contrast,sh RNA-Slfn5 transfection significantly increased the expression of MT1-MMP protein and m RNA compared with the control group(m RNA level:3.56±0.34 vs.0.98±0.05,n = 3,P <0.05;Protein level: 0.97±0.12 vs 1.67±0.07,n = 3,P<0.05).3.3.3 Influence of Slfn5 on repair of carotid artery injuryAfter EPCs were transfected with sh RNA-Slfn5,sh RNA-control,Ad-Slfn5 and Ad-control,they were transplanted into the rat model of carotid bulbous artery injury.At 14 days,Evans blue staining was used to detect the rate of re-endothelialization of injured vessels.The results showed that:Compared with the normal saline group,the reendothelialization rate of normal EPCs transplantation was higher,indicating that EPCs has the possibility of promoting vascular reendothelialization.(The saline group vs EPCs group,11.78±4.87% vs 49.03±2.26%,P < 0.01)(n=3);In the sh RNA-Slfn5 EPCs group,there was increased reendothelization(85.32±3.89% vs.50.12±4.12% in the sh RNA-control EPCs group,P < 0.01)(n=3).While the over-expression of Slfn5 was associated with a decreased rate of reendothelization(21.61±3.89% vs.51.18±3.89% in Ad-control EPCs group,P < 0.01)(n=3).He staining was used to measure the thickness of new intima.The results showed that the ratio of new intima area to media area(I/M)of rats treated with normal EPCs was significantly lower than that of the normal saline group(0.843±3.17 vs 0.524±2.87 in the saline group vs EPCs group;N =9,P < 0.05).Intracavascular I/M ratio was significantly reduced in the sh RNA-Slfn5 EPCs group(0.225±1.09 vs.0.518±2.31 in the sh RNA-Slfn5 EPCs group;n = 9;P < 0.05);While the overexpression of Slfn5 showed more intimal hyperplasia(0.923±1.89% vs.0.511±2.77,P < 0.01)in the Ad-Slfn5 EPCs group versus the Ad-control EPCs group(n=9).4 Conclusion4.1 Slfn5 is highly expressed in spleen;4.2 Silencing of Slfn5 expression by RNA interference significantly promoted the proliferation,migration,re-endothelialization ability of EPCs and the ability of new intima regeneration.On the contrary,overexpression of Slfn5 inhibited the ability of EPCs to proliferate,migrate,re-endothelialize and regenerate new intima;4.3 MT1-MMP is the downstream target of Slfn5 action.