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Erschienen in:
22.01.2021 | Sleep Breathing Physiology and Disorders • Original Article
Intermittent hypoxia-induced autophagy via AMPK/mTOR signaling pathway attenuates endothelial apoptosis and dysfunction in vitro
Erschienen in: Sleep and Breathing | Ausgabe 4/2021
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Purpose
The aim of this study was to examine whether or not intermittent hypoxia (IH) upregulated autophagy and the contributions of autophagy to endothelial apoptosis and dysfunction in human umbilical vein endothelial cells (HUVECs).
Method
HUVECs were incubated under normoxia and IH conditions. After 3-, 6-, 12-, and 24-h exposure, the autophagic vacuoles and autophagosomes were observed by transmission electron microscopy and monodansylcadaverine staining. The protein levels of autophagy-related biomarkers and AMPK/mTOR pathway were measured by Western blot. The apoptosis-related proteins and the percentage of apoptotic cells were evaluated by Western blot and flow cytometry, respectively, while the levels of endothelial function biomarkers were assessed by ELISA.
Results
IH induced autophagy, as determined by the increased numbers of the autophagic vacuoles, autophagosomes, and by the elevated levels of Beclin-1 protein, the LC3II/LC3I ratio, and p62 degradation. IH-induced autophagic flux peaked at 12-h duration and weakened at 24 h. IH increased the ratio of p-AMPK/AMPK and decreased the ratio of p-mTOR/mTOR, while compound C restored the alteration. A significant decrease in the Bcl-2 level and the Bcl-2/Bax ratio and a significant increase in the protein expression levels of Bax and cleaved caspase 3 and in the percentage of apoptosis were observed under IH exposure. Moreover, the NO level was reduced, while the ET-1 and VEGF levels were raised under IH condition. These alterations were suppressed by the pretreatment of 3-methyladenine.
Conclusions
IH upregulates autophagy through AMPK/mTOR pathway in HUVECs in vitro, which might be protective against endothelial apoptosis and dysfunction caused by IH.