Mechanism Of FoxO1 Affecting Autophagy In Diabetic Aortic Endothelial Cells

Objective:Establishing the methods of extracting and cultivating human aortic endothelial cells（HAECs）in vitro.Methods:Stripping endodermis from human aorta,using collagenaseⅠto digest endothelial cells from endodermis,and then utilizing immunohistochemistry and flow cytometry（FCM）to detect purity of HAECs.Results:0.1%and 0.2%collagenase type I,the best digestion time was 50min,2h adherent cells number were 2.52*10~4±2.10*10~3,4.05*10~4±3.84*10~3.After cell fusion,cells were cobblestone-like growth in inverted microscope.Cells were identified by CD31+andⅧfactors as positive,the cell purity reached 96.43%±4.12%（n=5）.The ability of endocytosis of ox-LDL and angiogenesis were normal.Conclusions:The method that blunt dissection of the aortic endothelium and Simply use Type I collagenase digestion using Type I collagenase digestion simply was better toseparate HAECs.Enzyme concentration and digestion time are important factors.The method is simple and convenient,shortening the training cycle and obtaining high purity cells.It is beneficial to the endothelial cells experiment in vitro.Aims: Inadequate autophagy contributed to endothelial dysfunction in diabetic patients.We aimed to investigate the relationship between inadequate autophagy and endothelial cells（ECs）apoptosis in diabetes and its underlying mechanism.Methods and Results: Aortic ECs were isolated from diabetic patients.Cultured human aortic ECs（HAECs）were stimulated with advanced glycation end products（AGEs）.The expression of autophagy and apoptosis-related proteins were determined by western blotting.Autophagosomes were observed by electron microscopy.The fusion of autophagosome and lysosomes was detected by immunofluorescence.Compared with non-diabetic subjects,the levels of LC3-Ⅱ and p-62 were markedly increased in ECs from diabetic patients,accompanied by the decreased expressions of Atg14,STX17 and co-localization of LC3-Ⅱ with LAMP2,and Atg14 with STX17.Long term stimulation of AGEs markedly upregulated LC3-II and p62 expression and the number of autophagosomes with decreased level of Atg14,STX17,Rab7 and co-localization of LC3-Ⅱ with LAMP2,and Atg14 with STX17 in HAECs.The apoptosis rates were significantly increased with elevated cleaved-caspase-3 level and declined Bcl-2 expression.Inhibition of autophagy with 3-MA could reduce AGEs-induced HAECs apoptosis.Higher levels of Fox O1,Ac-Fox O1 and Ac-Fox O1 binding to Atg7 were detected in AGEs-treated HAECs.Knockout Fox O1 by si Fox O1 reduced AGEs-induced autophagy and promoted the expression of Atg14 and the co-localization of LC3-Ⅱ with LAMP2,and Atg14 with STX17.Conclusions: Inadequate autophagy with impaired autophagosome-lysosomal fusion exists in ECs from diabetic patients.Fox O1 mediates AGEs-induced ECs autophagic apoptosis through impairing autophagosome-lysosomes fusion by inhibiting Atg14 expression.