Beclin-1/LC3-II dependent macroautophagy was uninfluenced in ischemia-challenged vascular endothelial cells

Abstract

Autophagy has been extensively studied and occurs in many biological settings. However, a question remains as to whether ischemia enhances Beclin-1/LC3-II-dependent macroautophagy in vascular endothelial cells, as has been previously thought. Furthermore, the effect of the level of autophagy on cell or skin flap survival still requires elucidation. We created a lethal ischemia model in human umbilical vascular endothelial cells (HUVECs), performed quantitative proteomics and bioinformatics analyses, and verified the autophagic status and effect both in vitro and in vivo. The significantly upregulated proteins encoded by autophagy-related genes (ATGs) included ATG2A, ATG3, ATG4B, ATG5, ATG7, ATG9A, ATG12, ATG16, and ATG101. The significantly enhanced lysosomal proteins were cathepsin B, cathepsin D, lysosome-associated membrane protein 1 (LAMP1), and LAMP2. However, the differentially expressed proteins excluded Beclin-1, microtubule-associated protein light chain 3 (LC3)-I, and LC3-II. Western blot analyses verified that the protein expression levels of Beclin-1, LC3-I, and LC3-II were neither upregulated nor downregulated in ischemia-challenged HUVECs. The autophagic status was not enhanced by rapamycin in ischemic HUVECs but appeared to be inhibited by chloroquine. Our in vivo study on rats showed that a downregulation in autophagic status jeopardized skin flap survival. In conclusion, Ischemia neither enhanced nor inhibited Beclin-1/LC3-II-dependent canonical macroautophagy both in vitro and in vivo, in contradiction to previous studies. An appropriate autophagic homeostasis can minimize cell or skin flap damage.

Keywords

• Autophagy
• Bioinformatics analysis
• Human umbilical vein endothelial cell
• Quantitative proteomics study
• Skin flap