Biological Effects And Mechanism Of Liraglutide On High Glucose Induced Human Endothelial Colony Forming Cells

Background and ObjectiveIn recent years,the incidence of diabetes continues to rise,and diabetic vascular complications have been a serious threat to the quality of life of patients with diabetes.Previous studies have found that it had a broad therapeutic prospect for the treatment and improvement of diabetic vascular complications by regulating stem cells,replacing or repairing damaged cells,or altering the biological behavior of cells.Endothelial colony forming cells(ECFC),as a subtype of endothelial progenitor cells(EPCs),play an important role in repairing vascular injury and promoting angiogenesis.However,ECFC's ability of proliferation,migration,and tube formation would be impaired to a different degree in the case of high glucose,and the specific mechanism remain unclear.The latest study shows that Liraglutide can significantly reduce the risk of cardiovascular diseases in patients with type 2 diabetes.However,its mechanism beyond the scope of slightly improving metabolic effect remains to be further studied.Therefore,we choose ECFC,which has more powerful biological activity according to recent studies,as target and to build a high-glucose model in vitro.Then we observe the proliferation,senescence,migration and tube formation of ECFC as well as changes in protein expression of sirtuins,vascular endothelial growth factor(VEGF)and Angiogen.Finally we added appropriate concentration of Liraglutide to intervene to explore whether it can regulate the proliferation,senescence,migration,and tube formation of ECFC through upregulating protein expression of Sirt1,VEGF and Angiogenin.MethodsFirstly,the peripheral blood of healthy adults was collected,and mononuclear cells(MNCs)were isolated from the human peripheral blood by density gradient centrifugation.ECFC was induced and subcultured by the EBM-2 complete culture medium.The cells were divided into three groups,normal glucose group(NG 5.5mmol/L D-glucose×6D),high glucose group(HG 30mmol/L D-glucose×6D),and osmotic pressure group(OSM 5.5mmol/L D-glucose+24.5mmol/L mannitol×6D).The proliferation,senescence,migration and tube formation of the three groups were tested respectively by the method of EdU experiment,SA-?-gal,Transwell experiment and tube formation experiment.The protein expression levels of Sirtuins(Sirt1-Sirt7),VEGF and Angiogenin were measured by Western blot.Then the concentration gradient of the Liraglutide(0,25,50,75,100,125nmol/L Liraglutide)was explored by the Western blot in high glucose environment(30mmol/LD-glucose).After obtaining the optimal concentration of Liraglutide intervention,the cells were divided into four groups,normal glucose group(NG 5.5mmol/L D-glucose ×6d),normal glucose intervention group(NG + Lira 5.5mmol/L D-glucose + 100nmol/L Liraglutide ×6d),high glucose group(HG 30mmol/L D-glucose ×6d),and high glucose intervention group(HG + Lira 30mmol/L D-glucose + 100nmol/L Liraglutide ×6d)in order to observe the effect of Liraglutide on the protein expression of VEGF and Angiogenin and the proliferation,senescence,migration and tube formation.Results1.Compared with the normal glucose control(5.5mmol/L D-glucose)ECFC' ability of proliferation,migration and tube formation have decreased while aging rate increased in high glucose group(30mmol/L D-glucose).The differences are statistically significant.And the changes are independent of the elevated osmotic concentration.2.Compared with the normal glucose group(5.5mmol/L D-glucose),the protein expression level of Sirtuins has decreased in high glucose group(30mmol/L D-glucose).Among them,the differences of Sirt1,Sirt2,Sirt7 are statistically significant,the protein expressions of the VEGF and Angiogenin are significantly decreased and the differences are statistically significant.The changes are also independent of the elevated osmotic concentration.3.After the ECFC intervened by the high glucose(30mmol/L D-glucose)+(0,25,50,75,100,125nmol/L Liraglutide),the protein expression level of the Sirt1 increases with the increase of the Liraglutide concentration.It starts to be statistically significant at 50 nmol/L and it reaches a plateau at 100nmol/L.4.Compared with the high glucose group(30mmol/L D-glucose),the protein expressions of the VEGF and the Angiogenin have increased in the high glucose intervention group(30mmol/L D-glucose + 100nmol/L Liraglutide).The differences are statistically significant.Meanwhile,ECFC' ability of proliferation,migration and tube formation have improved while the aging rate decreased.The differences are statistically significant.However,In the case of the normal glucose(5.5 mmol/L D-glucose),Liraglutide does not have this effect.Conclusion1.High glucose may accelerate cell aging while decrease the ability of proliferation,migration,and tube formation in ECFC.2.Whereas the expression of the Sirtuins,VEGF and Angiogenin were strongly down-regulated,concomitant with impairment of angiogenesis in ECFC exposed to high glucose.3.Liraglutide can prevent high glucose–induced cell aging phenotype,and increase its ability of proliferation,migration,tube formation,partly through up-regulating the protein expression of the Sirt1,VEGF and Angiogenin.