VEGF Promotes Osteogenic Differentiation Of BMSCs Co-cultured With EPCs Via YAP Signaling Pathway

Background: Bone tissue engineering is a promising therapeutic strategy to promote bone regeneration.Bone marrow mesenchymal stem cells(BMSCs)and endothelial progenitor cells(EPCs)are commonly used cells for promoting osteogenesis and angiogenesis in tissue engineering.High glucose environment can affect BMSCs cell function,and BMSCs combined with EPCs could improve bone formation in vivo.While,its potential mechanism is unclear.Paracrine is one of the mechanisms for cell-cell interaction.Vascular endothelial growth factor(VEGF)can be secreted by various cells and plays a crucial role in all stages of bone repair,including inflammation,intramembranous and endochondral ossification.It is one of the most useful growth factors to induce angiogenesis and vascularization in bone repair and regeneration.Purpose: The purpose of this experimental study is to investigate the expression changes of VEGF in the co-culture system of BMSCs and EPCs,and its effects on the proliferation,migration,and osteogenic differentiation of BMSCs in high glucose environment,as well as the related mechanism.Methods:1.Using density gradient centrifugation combined with differential attachment method separated primary rat BMSCs and EPCs.The obtained cells were identified by induction differentiation experiment or tubule formation experiment,combined with cell morphology and cell surface markers.2.Medium was prepared to culture BMSCs containing different concentrations of VEGF in high glucose environment.The cell proliferative activity was detected by CCK8 method,and selected the appropriate concentration.The BMSCs were treated with VEGF,and the ALP staining and RT-PCR were used to detect the osteogenic differentiation of BMSCs.The scratch test and Transwell migration test were conducted to determine their effects on the migration ability of BMSCs.3.After VEGF was applied to BMSCs,the mRNA levels of VEGFR1,VEGFR2 and VEGFR3 were detected by RT-PCR.The changes of cytoskeletal protein F-actin and nuclear translocation of YAP protein were detected by immunofluorescence,and the total content of YAP protein was detected by western blot.Results:1.Primary rat BMSCs and EPCs were successfully isolated by the above methodes.The induction and differentiation experiments showed that BMSCs had adipogenic,osteogenic and chondrogenic differentiation potential,and the cell surface markers showed CD90+,CD44+,CD31-,and CD45-;The tubular formation experiment showed that EPCs had angiogenic function,and cell surface markers showed CD31+,CD144+,and VEGFR2+.2.The results of CCK8 showed that when the concentration of VEGF was higher than 2.5ng/m L in high glucose environment,the proliferative activity of BMSCs was significantly increased,so the concentration of2.5ng/m L was selected for the subsequent experiment.Seven days after osteogenic induction,the cells in the VEGF group were deeply stained with ALP and the RT-PCR assays showed up-regulation of OCN,Runx2 and Osterix gene expressions.After BMSCs incubated with VEGF for 24 h,the residual rate of scratched surface was 9.7%±0.6%,which was significantly lower than that in the BMSCs control group(19.3% ±1.3%).The number of migrated cells in the BMSCs +VEGF group in the transwell migration test was more than that in the other two groups,which was consistent with the results of the scratch test.3.After VEGF was applied to BMSCs,T-PCR showed that the expression of VEGFR2 was up-regulated(P<0.05),and the use of VEGFR2 receptor inhibitor could reverse the enhancement of VEGF on osteogenic differentiation of BMSCs.Immunofluorescence staining showed that the morphology and quantity of cytoskeletal protein F-actin were significantly changed,and the nuclear transfer of YAP protein was significantly increased.The results of western blot experiment also showed that the total content of YAP protein was increased.Conclusions:1.Primary BMSCs and EPCs can be successfully separated and cultured by density gradient centrifugation combined with differential attachment method.2.VEGF can enhance the proliferative activity,migration and osteogenic differentiation of BMSCs in high glucose environment.3.VEGF acts on cells through VEGFR2 receptor,which may affect cell function by remodeling cytoskeletal protein F-actin and regulating the nuclear transfer amount of YAP protein.