ETV2 Overexpression Regulates Osteogenic And Angiogenic Differentiation Of Human Dental Pulp Stem Cells

Background and ObjectivesHow to effectively repair bone defects in a short time has plagued clinicians for a long time and is urgent to be solved.There are many possible causes of bone defects in the oral and maxillofacial region,including tumors,trauma inflammation,and congenital malformations.The bone defects occurring in the oral and maxillofacial region may bring inconvenience to the patients’ life.They may affect the patients’pronunciation,swallowing or chewing.Also,they could damage the patients’ mental health and hinder the patients’ normal social activities.How to repair bone defects quickly and effectively has been reported by many researches.There are many drawbacks in the traditional methods of repairing bone defect,such as secondary injury to patients,a long duration of repair,and poor quality of in-duced new bone formation.Thus,it is difficult for them to be widely applied.With the development of tissue engineering technology,it provides new insights into repair of oral and maxillofacial bone defects.To construct tissue-engineered bone successfully,it is paramount to choose the appropriate seed cells.Embryonic stem cells and in-duced pluripotent stem cells have shown strong abilities of cell proliferation and mul-tilineage differentiation.Yet,they are difficult to be widely accepted as seed cells considering ethical issues and potential tumorigenicity.Mesenchymal stem cells(MSCs)have the potential for broad application in the field of tissue engineering be-cause of a variety of sources and good ability of multilineage differentiation.MSCs extracted from human dental pulp tissue,namely human dental pulp stem cells(hDPSCs),can be used as one of the ideal seed cells for tissue engineering.In view of the low survival rates of seed cells after being transplanted,it is im-portant to enhance the ability of osteogenic differentiation of seed cells to guarantee the efficacy of bone defect repair.In previous studies,to improve bone regeneration in tissue engineering,many diferent gene vectors have been used to transfer exogenous genes that can promote osteogenesis into seed cells.However,it is not enough to im-prove the potential of osteogenic differentiation of seed cells alone,considering that vascularization also plays an important role in bone regeneration in tissue engineering.Without enough vascularization in the bone defect site,the survival of seed cells will be threatened,which can eventually lead to the failure of repairing the bone defects by tissue-engineered bone.Therefore,it is necessary to enhance osteogenesis and angio-genesis of seed cells simultaneously to improve bone regeneration and vascularization in the cell implantation sites.Ets variant 2(ETV2),also known as ER71/Etsrp,be-longs to the Ets protein family.Previous studies have shown that ETV2 is closely re-lated to angiogenesis.ETV2 alone or in combination with other transcription factors,can induce different types of cells into vascular endothelial cells.However,few stud-ies about the relationship between ETV2 and osteogenic differentiation have been re-ported.hDPSCs were selected as seed cells and this study aims to investigate the regu-latory effect of ETV2 on osteogenic and angiogenic differentiation of hDPSCs,and to explore the possible mechanism.In this study,we investigated the effect of ETV2 overexpression on the angiogenic differentiation of hDPSCs by in vitro and in vivo experiments at first,and then explored the underlying mechanisms through proteomic analysis.At the same time,a series of in vitro and in vivo assays were used to study the possible effect of inducible overexpression of ETV2 on osteogenic differentiation of hDPSCs.Also,transcriptome sequencing was used to unearth the underlying regu-latory mechanism.Altogether,this study aims to provide new avenues and more op-tions towards developing vascularized tissue-engineered bone.Methods1.Culture and identification of hDPSCs and in vitro studies on the effect of ETV2 overexpression on the angiogenic differentiation of hDPSCshDPSCs were isolated and cultured from the pulp tissue of the extracted teeth and purified by limiting dilution assay.Next,the colony forming potential of hDPSCs was determined by crystal violet staining.Additionally,flow cytometry was utilized to study the expression of cell surface markers CD24,CD29,CD34,CD44,CD45,CD73,and CD90.Meanwhile,the osteogenic,adipogenic and chondrogenic differen-tiation capacity of hDPSCs was detected by alizarin red staining,oil red O staining,and alcian blue staining.Afterwards,hDPSCs were infected by ETV2-overexpressing or control lentiviral particles and infection efficiency was evaluated by qRT-PCR,Western Blot analysis and immunofluorescence assay.Subsequently,CCK-8 assay was utilized to evaluate the influence of ETV2 overexpression on the proliferation ability of hDPSCs;qRT-PCR and Western Blot were performed to analyse the effect of ETV2 overexpression on the expression levels of angiogenesis-related factors CD31,VE Cadherin,VEGFR1 and VEGFR2 at the mRNA and protein levels at days 0,7 and 14 after angiogenic induction;immunofluorescence and flow cytometry were used to detect the effect of ETV2 overexpression on the expression level of VE Cad-herin at day 7 after angiogenic induction;and in vitro tubulogenesis assay was also used to analyze the effect of ETV2 overexpression on the tubulogenesis ability of hDPSCs.2.Proteomic analysis of the effect of ETV2 overexpression on angiogenic differentia-tion of hDPSCs and in vivo Matrigel plug assayhDPSCs infected with ETV2-overexpressing or control lentiviral particles were gathered after 10 days of angiogenic induction,and then proteomics analysis was performed.Differentially expressed proteins were selected according to the specified inclusion criteria and then were used to generate the heatmap.Bioinformatic analysis of the differentially expressed proteins was performed using the Geneontology(GO)database,the Kyoto Encyclopedia of Genes and Genomes(KEGG)database and the Reactome database.To explore whether ETV2 overexpression can promote angio-genic differentiation in vivo,Matrigel plug assay on nude mice was performed.Then,gross observation,Haematoxylin&Eosin(HE)staining,and CD31 immunohisto-chemical staining were used to detect the effect of ETV2 overexpression in hDPSCs on angiogenesis in vivo.3.In vitro studies on the effect of inducible overexpression of ETV2 on osteogenic differentiation of hDPSCsLentiviral vectors based on the tetracycline-regulated gene expression system were used for inducible overexpression of ETV2 in hDPSCs,and the optimal concen-tration of doxycycline(Dox),which induces gene expression,was screened.Accord-ing to the presence or absence of Dox and different types of lentiviral vectors,the hDPSCs were divided into four groups:Tet-on-NC-Dox(-),Tet-on-NC-Dox(+),Tet-on-ETV2-Dox(-),and Tet-on-ETV2-Dox(+).qRT-PCR,Western Blot and im-munofluorescence were used to detect the infection efficiency.Subsequently,CCK-8 assay was used to detect the effect of inducible overexpression of ETV2 on the prolif-eration of hDPSCs;qRT-PCR and Western Blot were used to investigate the effect of inducible overexpression of ETV2 on the expression of osteogenesis-related factors ALP,COL1 A1,and OSX at the mRNA and protein levels at days 3,7 and 14 after osteogenic induction;immunofluorescence was used to detect the effect of inducible overexpression of ETV2 on the expression level of osteogenesis-related factor OSX at day 3 after osteogenic induction;ALP staining and measurement of ALP activity were conducted on day 7 and day 14 after osteogenic induction;alizarin red staining and detection of calcium concentration were conducted on day 14 and day 21 after osteo-genic induction.4.RNA-sequencing analysis on the mechanism of the promoting effect of inducible overexpression of ETV2 on osteogenic differentiation of hDPSCshDPSCs isolated from 3 different donors were infected with Tet-on-ETV2 lenti-viral vector.On day 10 after osteogenic induction with or without Dox,total RNA was extracted and RNA-sequencing analysis was performed.Differentially expressed genes were selected according to the specified inclusion criteria and then the heatmap was generated.Three up-regulated genes and three down-regulated genes were se-lected to verify the accuracy of RNA-sequencing using qRT-PCR.Subsequently,the GO database and KEGG database were used to perform bioinformatics analysis of the differentially expressed genes.The possible signaling pathways involved in the pro-moting effect of ETV2 on osteogenic differentiation were chosen,followed by con-firmation by Western Blot.qRT-PCR,Western Blot and immunofluorescence were conducted to assess the effects of adding inhibitors of specific pathways on the ex-pression levels of osteogenesis-related factors ALP,COL1A1 and OSX at the mRNA or protein level on day 3 after osteogenic induction.ALP staining and measurement of ALP activity were performed on day 7 and day 14 and alizarin red staining and detec-tion of calcium concentration were conducted at day 14 and day 21 after osteogenic induction to explore the effect of adding inhibitors of specific pathways on ALP avtivity and formation of mineralized nodule.5.In vivo studies on the effect of inducible overexpression of ETV2 on new bone formationhDPSCs infected with Tet-on-NC or Tet-on-ETV2 lentivirus received osteogenic induction in the presence or absence of Dox.On day 7 after osteogenic induction,hDPSCs were trypsinized and the cell suspensions were added to the β-TCP.Then,hDPSCs/β-TCP composites were used in rat skull defect experiment and subcutane-ous ectopic osteogenesis assay in nude mice.Based on different components in the composites,rats or mice in this study can be divided into four groups:Tet-on-NC-Dox(-)group,Tet-on-NC-Dox(+)group,Tet-on-ETV2-Dox(-)group and Tet-on-ETV2-Dox(+)group.8 weeks later,the rats were euthanized,and the skull specimens were used for micro-CT scanning.HE staining and Masson staining were conducted after decalcification of the skull specimens.Also,the samples transplanted subcutaneously in nude mice were taken out for HE staining and Masson staining af-ter being decalcified.Results1.Culture and identification of hDPSCs and the effect of ETV2 overexpression on the angiogenic differentiation of hDPSCsThe results showed that hDPSCs presented fibroblast-like spindle morphology,colony forming ability and multilineage differentiation potential.Also,the expression of cell surface markers of hDPSCs was in line with the characteristics of MSCs.The results of qRT-PCR,Western Blot,and immunofluorescence showed that the mRNA and protein levels of ETV2 were significantly upregulated after lentiviral infection.The result of CCK-8 assay revealed that compared with the control group,the prolif-eration of hDPSCs in the ETV2-overexpressing group was significantly inhibited.On days 0,7,and 14 after angiogenic induction,results of qRT-PCR and Western Blot showed that the expression levels of angiogenesis-related factors(CD31,VE Cadher-in,VEGFR1,and VEGFR2)at the mRNA and protein levels in the ETV2-overexpressing group were significantly higher than those in the control group;on day 7 after angiogenic induction,results of immunofluorescence and flow cytome-try showed that the expression level of VE Cadherin in the ETV2-overexpressing group were significantly increased compared with that in the control group;mean-while,results of in vitro tubulogenesis assay showed that the tubule length and the number of branch points in the ETV2-overexpressing group were significantly en-hanced compared to those in the control group.2.Results of proteomic analysis after ETV2 overexpression and results of in vivo Matrigel plug assayThe results of proteomics showed that 941 proteins were up-regulated and 883 proteins were down-regulated in hDPSCs in the ETV2-overexpressing group in com-parison with the control group.The results of bioinformatics analysis showed that the differentially expressed proteins expressed differential proteins were mainly enriched in biological processes or molecular functions related to biological oxidation,energy metabolism,mitosis,and nucleic acid metabolism.Meanwhile,the results of pathway enrichment analysis revealed that up-regulated proteins could be enriched to the VEGF pathway.The results of Matrigel plug assay showed that the Matrigel plugs in ETV2-overexpressing group(ETV2 group)and HUVEC s group presented light red with visible microvessels by gross observation,while the Matrigel plugs in the Blank group and the Control group was almost transparent.The results of HE staining showed that the number of microvessels in the ETV2 group or HUVECs group was significantly higher than that in the Blank or Control group.The results of CD31 im-munofluorescence staining showed that there was little or no positive staining of CD31 in Blank or Control group.In comparison,the CD31-positive staining area was significantly increased in the ETV2 or HUVECs groups.3.Inducible overexpression of ETV2 can promote osteogenic differentiation of hu-man hDPSCsAfter hDPSCs were infected by ientiviral vectors based on the tetracy-cline-regulated gene expression system,the optimal concentration of Dox was deter-mined to be 100 ng/mL.Meanwhile,the results of qRT-PCR,Western Blot and im-munofluorescence assays confirmed the mRNA and protein levels of ETV2 were sig-nificantly upregulated after Dox induction.Subsequently,the results of CCK-8 showed that no significant difference was found among the groups.On days 3,7,and 14 after osteogenic induction,results of qRT-PCR and Western Blot showed that the mRNA and protein levels of osteogenesis-related factors(ALP,COL1A1 and OSX)in the Tet-on-ETV2-Dox(+)group were significantly higher than those in the other three groups;on day 3 after osteogenic induction,the results of immunofluorescence assay showed that the expression level of OSX in the Tet-on-ETV2-Dox(+)group was sig-nificantly increased compared with that in the other three groups;the results of ALP staining and activity assay on days 7 and 14 after osteogenic induction,and the results of alizarin red staining and detection of calcium concentration on days 14 and 21 after osteogenic induction showed that the Tet-on-ETV2-Dox(+)group showed signifi-cantly stronger ALP activity and ability of calcium deposition than the other three groups.4.Results of transcriptome sequencing analysis and mechanisms of inducible overex-pression of ETV2 promoting osteogenic differentiation of hDPSCsThe results of transcriptome sequencing showed that 732 genes were up-regulated and 389 genes were down-regulated in ETV2-overexpressing group compared with the control group.Subsequently,the results of qRT-PCR confirmed the accuracy of sequencing results.The results of bioinformatics analysis showed that among 1121 differentially expressed genes,626 genes were annotated to specific GO terms,and the top 25 GO terms included positive regulation of ERK1 and ERK2 cas-cades,suggesting that the ERK1/2 MAPK pathway may be involved in the regulation of osteogenic differentiation by ETV2 overexpression.There were 274 genes mapped to specific KEGG pathways,and the top 25 KEGG pathways included the PI3K-Akt pathway known to be closely related to osteogenic differentiation.The results of Western Blot showed that the phosphorylation levels of ERK protein and AKT protein in ETV2-overexpressing hDPSCs were significantly increased compared with those in control cells.The results of qRT-PCR,Western Blot and immunofluorescence demon-strated that the phosphorylation levels of ERK protein and AKT protein,and the mRNA and protein levels of osteogenesis-related factors(ALP,COL1A1 and OSX)were significantly increased in the inducible ETV2-overexpressing group compared with the control group on day 3 after osteogenic induction.However,the elevated phosphorylation level of the pathway proteins,and enhances the mRNA and protein expression levels of osteogenesis-related factors,were partially decreased after the application of ERK 1/2 MAPK pathway inhibitors or PI3K-Akt pathway inhibitors.Meanwhile,the results of ALP staining and activity assay,and alizarin red staining and detection of calcium concentration also showed the same trend.5.Results of in vivo study on the effect of inducible overexpression of ETV2 on new bone formationThe results of rat skull defect experiment revealed that after micro-CT scanning,the bone mineral density(BMD),relative bone volume(BV/TV)and bone thickness(Tb.Th)in the Tet-on-ETV2-Dox(+)group were significantly higher than those in the other three groups.The results of HE staining and Masson staining of rat skull bones presented that more new bone formation was found in the Tet-on-ETV2-Dox(+)group when compared with the other three groups.The results of HE staining and Masson staining of the samples extracted subcutaneously from the nude mice showed the same tendency.Conclusions1.ETV2 overexpression possesses the potential to promote the angiogenic differentia-tion of hDPSCs in vitro,and VEGF pathway is involved in the promoting effect.2.ETV2 overexpression can enchance the blood vessel formation of hDPSCs in Mat-rigel plug assay.3.Inducible overexpression of ETV2 can promote the osteogenic differentiation of hDPSCs in vitro,and ERK1/2 MAPK pathway and PI3K-Akt pathway are involved in the promoting effect.4.The hDPSCs/β-TCP composites containing inducible ETV2-overexpressing hDP-SCs increase bone formation in rat skull defect assay and subcutaneous ectopic oste-ogenesis assay in nude mice.