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 intima and ECs were isolated from diabetic patients. Cultured human aortic endothelial cells (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, p62, FoxO1, and Ac-FoxO1 were increased in ECs from diabetic patients, accompanied by the decreased expressions of Atg14, STX17, and co-localization of LC3-II/LAMP2 and Atg14/STX17. Long-term stimulation with AGEs up-regulated LC3-II and p62 expression and the number of autophagosomes with decreased level of Atg14, STX17, Ras-related protein 7 (Rab7), and co-localization of LC3-II/LAMP2 and Atg14/STX17 in HAECs. The apoptosis rates were increased with elevated cleaved-caspase-3 and declined Bcl-2 expression. Inhibition of autophagy with 3-methyladenine could reduce long-term AGEs-induced apoptosis. Higher levels of FoxO1, Ac-FoxO1, and Ac-FoxO1 binding to Atg7 were detected in AGEs-treated HAECs. AGEs-induced FoxO1 enhanced Akt activity, decreased SIRT1-deacetylase activity by phosphorylation and elevated Ac-FoxO1. Knockout of FoxO1 reduced AGEs-induced autophagy and promoted the expression of Atg14 and the co-localization of LC3-II/LAMP 2 and Atg14/STX17. CONCLUSION Inadequate autophagy with impaired autophagosome-lysosomal fusion exists in aortic intima and ECs from diabetic patients. FoxO1 mediates AGEs-induced ECs autophagic apoptosis through impairing autophagosome-lysosomes fusion by inhibiting Atg14 expression.

中文翻译：

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FoxO1抑​​制自噬小体-溶酶体融合，导致糖尿病中的内皮自噬细胞凋亡。

AIMS自噬不足导致糖尿病患者的内皮功能异常。我们旨在研究糖尿病中自噬不足与内皮细胞（ECs）凋亡之间的关系及其潜在机制。方法和结果从糖尿病患者中分离出主动脉内膜和ECs。培养的人主动脉内皮细胞（HAEC）用高级糖基化终末产物（AGEs）刺激。通过蛋白质印迹法测定自噬和凋亡相关蛋白的表达。通过电子显微镜观察自噬体。通过免疫荧光检测自噬体和溶酶体的融合。与非糖尿病患者相比，糖尿病患者的EC中LC3-II，p62，FoxO1和Ac-FoxO1的水平升高，同时Atg14，STX17，和LC3-II / LAMP2和Atg14 / STX17的共定位。AGEs的长期刺激上调了LC3-II和p62的表达以及Atg14，STX17，Ras相关蛋白7（Rab7）水平降低以及LC3-II / LAMP2和Atg14 / HAEC中的STX17。裂解的caspase-3水平升高，凋亡率增加，Bcl-2表达降低。3-甲基腺嘌呤抑制自噬可以减少AGEs诱导的长期凋亡。在AGEs处理的HAEC中检测到更高水平的FoxO1，Ac-FoxO1和Ac-FoxO1与Atg7的结合。AGEs诱导的FoxO1通过磷酸化增强了Akt活性，降低了SIRT1-deacetylase活性，并增加了Ac-FoxO1。FoxO1的基因敲除减少了AGEs诱导的自噬，并促进了Atg14的表达以及LC3-II / LAMP 2和Atg14 / STX17的共定位。结论糖尿病患者的主动脉内膜和EC存在自噬不足和自噬体-溶酶体融合受损。FoxO1通过抑制Atg14的表达来破坏自噬体与溶酶体的融合，从而介导AGEs诱导的ECs自噬凋亡。