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.

自噬已被广泛研究并发生在许多生物环境中。然而，一个问题仍然存在，即缺血是否会增强血管内皮细胞中 Beclin-1/LC3-II 依赖性巨自噬，正如之前所认为的那样。此外，自噬水平对细胞或皮瓣存活的影响仍有待阐明。我们在人脐带血管内皮细胞 (HUVEC) 中创建了致死性缺血模型，进行了定量蛋白质组学和生物信息学分析，并在体外和体内验证了自噬状态和效果。自噬相关基因 (ATG) 编码的显着上调蛋白包括ATG2A、ATG3、ATG4B、ATG5、ATG7、ATG9A、ATG12、ATG16和ATG101。显着增强的溶酶体蛋白是组织蛋白酶 B、组织蛋白酶 D、溶酶体相关膜蛋白 1 (LAMP1) 和 LAMP2。然而，差异表达的蛋白质排除了 Beclin-1、微管相关蛋白轻链 3 (LC3)-I 和 LC3-II。蛋白质印迹分析证实，Beclin-1、LC3-I 和 LC3-II 的蛋白质表达水平在缺血挑战的 HUVEC 中既没有上调也没有下调。雷帕霉素在缺血性 HUVEC 中没有增强自噬状态，但似乎被氯喹抑制。我们的体内对大鼠的研究表明，自噬状态的下调会危及皮瓣的存活。总之，缺血在体外和体内既没有增强也没有抑制 Beclin-1/LC3-II 依赖的经典巨自噬，这与之前的研究相矛盾。适当的自噬稳态可以最大限度地减少细胞或皮瓣损伤。