Id1 Regulates Cell Cycle Of Endothelial Progenitor Cells Through Inducing Wnt2 Expression

Background and ObjectiveOne of the start factors of atherosclerosis is vascular endothelial injury.Therefore,maintaining the structure and function of vascular endothelial cell layer is crucial to prevention and treatment of atherosclerosis.Endothelial progenitor cells(EPCs)is a kind of endothelial precursor cells,which was released into the peripheral blood followed by homing to the sites of vascular endothelial injury for re-endothelialization.However,the amount of EPCs in the peripheral blood is extremely limited,and the limited quantity is an important factor which restricts the function of EPCs in the repair of endothelial injury.Therefore,increasing the peripheral blood content of EPCs has become a hot research topic.In our previous studies,we found that Inhibitor of DNA binding-1(Idl)plays an important role in promoting the proliferation of EPCs.However,the specific mechanisms of Id1 promoting the proliferation of EPCs are not fully understood.Idl is a member of helix-loop-helix(HLH)family,and contributes to the process of cell proliferation,migration and other cellular activities.In tumor cells,Id1 can promote the expression of cell cycle regulatory factor Cyclin D1 to promote cell cycle progression from G1 phase to S phase.Cell cycle is closely related to cell proliferation,and whether Idl regulates EPCs cell cycle is not still documented.In addition,studies found that Wnt2 significantly reduced in the liver blood sinus endothelial cells of Idl-/-mice,and exogenous Wnt2 can restore the function of the liver vascular regeneration.Wnt2 plays an important role in angiogenesis and the differentiation of stem cells into vascular endothelial cells.Cyclin D1 is the classic downstream target of Wnt/β-Catenin signaling pathway,so we propose that Id1 can act on EPCs cell cycle regulation by Wnt2.This study explored the role of Idl in cell cycle regulation of EPCs and its possible mechanisms.It provides a new way to study the molecular mechanism of Id1 regulation of EPCs proliferation.methods1.Isolation and identification of EPCs1.1 Isolation and culture of mouse bone marrow derived EPCsBone marrow derived mononuclear cells were extracted from the tibia and femur of mice by gradient centrifugation,and then were inoculated into the culture flask of gelatin.The DMEM-F12 medium containing 20%fetal bovine serum was used for induced culture.After 48 hours of culture,the non-adherent cells were discarded.Fresh medium was given to the adherent cells for culture of another 4-7 days.1.2 Identification of EPCs1.2.1 The morphological characteristics of EPCs were observed under the inverted microscope.1.2.2 The cells were stained with Dil-Ac-LDL and FITC-UEA-1 and DAPI.And then,the confocal fluorescence microscope were used to detect the fluorescent staining rate.1.2.3 The cell phenotype was detected by flow cytometry with anti-mouse Sca-1 and VEGFR2 antibody.2.Effects of Id1 on cell cycle of EPCs2.1 Effects of overexpression of exogenous Id1 on the cell cycle of EPCs2.1.1 Recombinant adenovirus expressing Id1 was transfected into EPCs,and the transfection efficiency was detected by RT-PCR and Western blot.2.1.2 The cell cycle distribution of the EPCs was tested with Flow cytometry.2.1.3 The expression of Cyclin D1 was monitored using RT-PCR and Western blot.2.2 Effects of interfering expression of endogenous Id1 on the cell cycle of EPCs2.2.1 siRNA was used to transfected into EPCs to inhibit endogenous Id1 expression,and the interference efficiency was detected with RT-PCR and Western blot.2.2.2 Flow cytometry was used to detect the cell cycle distribution of the cells.2.2.3 The expression of Cyclin D1 was tested using RT-PCR and Western blot.3.The role of Wnt2 in the regulation of cell cycle by Id1 in EPCs.3.1 Regulation of exogenous Id1 on Wnt2 expression level and nuclear transfer ofβ-CateninThe total RNA,total protein,cytoplasmic protein and nuclear protein of Ad-transfected EPCs were extracted.The expression of Wnt2 and the content of β-Catenin in cytoplasm and nucleus were detected using RT-PCR or Western blot.3.2 Effects of interfering Id1 expression on the expression of Wnt2 and nuclear transfer of β-CateninThe total RNA,total protein,cytoplasmic protein and nuclear protein of siRNA-transfected EPCs were extracted.The expression of Wnt2 and the content of β-Catenin in cytoplasm and nucleus were tested using RT-PCR or Western blot.3.3 Effects of interfering Wnt2 expression on Id1 regulation of EPCs cell cyclesiRNA was transfected into Ad-Id1-infected EPCs to interfere Wnt2 expression.Flow cytometry was used to test the cell cycle distribution of the cells.The expression level of Id1,Wnt2,and Cyclin D1 were monitored using RT-PCR and Western blot.Results1.Effect of Id1 on cell cycle of EPCs1.1 Identification of mouse bone marrow derived EPCsBone marrow mononuclear cells cultured for 4-7 days were observed as large amount of fusiform,oval,spindle cells.Di1-Ac-LDL and FITC-UEA-1 double fluorescent staining identification:in the laser scanning confocal microscope visible,(91.4 + 2.0)%cells were both red and green fluorescent double staining.The double stained cells were considered as EPCs.Cell surface expression of Sca-1 and VEGFR-2 was detected by flow cytometry.The results showed that the positive rate of Sca-1 was 90.6%and the positive rate of VEGFR-2 was 94%.1.2 Effect of Id1 on cell cycle of EPCsOverexpression of exogenous Id1 promotes the evolution of EPCs cell cycleCompared with wild-type group and Ad-vector group,Ad-Id1 group was in a significant reduction in the number of cells in G1 phase and in a markedly increase in the number of cells in S/G2M phases.The difference is statistically significant(P<0.05).It suggested that Ad-Id1 can promote EPCs cell cycle from G1 phase into S/G2M.At the same time,mRNA and protein levels of Cyclin D1 were detected by RT-PCR and Western blot.Results indicate that compared with control group,the mRNA and protein levels of Cyclin D1 in Ad-Id1 group were markedly higher.1.3 Interfering endogenous Idl expression inhibits the evolution of EPCs cell cycleCompared with wild-type group and si-con group,si-Id1 group was in a significant increase in the number of cells in G1 phase and in a markedly reduction in the number of cells in S/G2M phases.The difference is statistically significant(P<0.05).It suggested that si-Id1 can inhibit EPCs cell cycle from G1 phase into S/G2M.At the same time,mRNA and protein levels of Cyclin D1 were detected by RT-PCR and Western blot.The results showed that the mRNA and protein levels of Cyclin D1 in si-Id1 group were markedly lower than those in both wild type group and si-con group.2.Id1 regulates EPCs cell cycle through up-regulation of Wnt2 expression2.1 Overexpression of exogenous Id1 promotes Wnt2 expression and nuclear transfer of p-CateninmRNA and protein levels of Wnt2 were detected using RT-PCR and Western blot.The results showed that mRNA and protein content of Wnt2 in Ad-Id1 group were much more compared with those in control group.Protein level of β-Catenin was detected using Western blot.According to the results,the content of β-Catenin in both cytoplasm and nucleus were much more in the Ad-Id1 group compared with control group.It suggested that Ad-Id1 could promote the expression of Wnt2 in EPCs,and promote the accumulation of β-Catenin in the cytoplasm and transfer to the nucleus.2.2 Interfering endogenous Id1 expression inhibits Wnt2 expression and nuclear transfer of β-CateninmRNA and protein levels of Wnt2 were detected using RT-PCR and Western blot.The results showed that mRNA and protein levels of Wnt2 in si-Id1 group were significantly lower than those in control group.Protein level of β-Catenin was detected using Western blot.The results showed that the content of β-Catenin in the cytoplasm and nucleus were remarkedly less in the si-Id1 group compared with si-con group and wild type group.It suggested that si-Idl could inhibit the expression of Wnt2 in EPCs,and inhibit the accumulation of β-Catenin in the cytoplasm and transfer to the nucleus.2.3 Interfering Wnt2 expression can blunt the EPCs cell cycle regulation of IdlThe proportion of cells in G1 phase in Ad-Id1/si-Wnt2 group was ramarkedly larger compared with that in Ad-Id1/si-con group,but smaller than that in Ad-vector/si-con group.There were significant differences(P<0.05).At the same time,the mRNA and protein levels of Cyclin D1 in Ad-Id1/si-Wnt2 group were both significantly lower than that in Ad-Id1/si-con group,but higher than that in Ad-vector/si-con group.It suggested that Wnt2 could partially block the regulation of Idl on cell cycle and Cyclin D1 expression in EPCs.ConclusionId1 can regulate EPCs cell cycle progression.Up-regulation of Wnt2 expression may be one of the molecular mechanisms of Id1 regulation of EPCs cell cycle progression.