Department of Physiology and Neuroscience, Soochow University - Dushu Lake Campus, Suzhou, China.
Department of Pharmacology and Laboratory of Aging and Nervous Diseases, Soochow University School of Pharmaceutical Science, Laboratory of Molecular Diagnostics, Medical College of Soochow University, Suzhou, P.R. China.
Clin Exp Hypertens. 2021 Jul 4;43(5):462-473. doi: 10.1080/10641963.2021.1901110. Epub 2021 Mar 29.
Signal transduction of Angiotensin II (Ang II) induced autophagy and its role in Ang II-induced dysfunction of HUVECs are still unclear.
HUVECs are stimulated with different doses of Ang II (10-9-10-5 mol/L) for different time (6-48 hours). Autophagy-related protein markers: LC3, Beclin-1 and SQSTM1/p62 are measured by western blot.
Incubation with Ang II increases autophagic flux (Beclin-1, autophagosomes formation, and degradation of SQSTM1/p62, LC3-I). Increased autophagic levels are inhibited by pretreatment with Ang II type 1 receptor (AT1) blocker (Candesartan), NADPH Oxidase inhibitor (apocycin), mitochondrial K channels inhibitor (5-hydroxydecanoate, 5HD). 3-Methyladenine (inhibitors of autophagy) and rapamycin (activator of autophagy) respectively inhibits or activates Ang II-induced autophagy levels. Ang II decreases phosphorylation of endothelial nitric oxide synthase (eNOS) and NO production in HUVECs. L-NAME (NOS inhibitor) totally mimics the actions of Ang II on eNOS, NO production and autophagy levels. Rapamycin further decreases NO production combined with Ang II. Silence Atg5 completely reverses Ang II-activated autophagy levels.
Our results demonstrate that Ang II stimulation increases autophagy levels via AT1 receptor, NADPH oxidase, mitochondrial K channel, eNOS, Atg5 signal pathway in HUVECs, and activation of autophagy contributes to Ang II induced dysfunction of HUVECs.
血管紧张素 II（Ang II）诱导的信号转导和自噬及其在 Ang II 诱导的 HUVECs 功能障碍中的作用尚不清楚。
用不同剂量的 Ang II（10-9-10-5 mol/L）刺激 HUVECs 不同时间（6-48 小时）。用 Western blot 法测定自噬相关蛋白标志物：LC3、Beclin-1 和 SQSTM1/p62。
Ang II 孵育增加自噬通量（Beclin-1、自噬体形成和 SQSTM1/p62、LC3-I 降解）。用 Ang II 型 1 受体（AT1）阻滞剂（坎地沙坦）、NADPH 氧化酶抑制剂（脱辅基细胞色素 c）、线粒体 K 通道抑制剂（5-羟基癸酸，5HD）预处理可抑制自噬水平增加。3-甲基腺嘌呤（自噬抑制剂）和雷帕霉素（自噬激活剂）分别抑制或激活 Ang II 诱导的自噬水平。Ang II 降低 HUVECs 内皮型一氧化氮合酶（eNOS）的磷酸化和 NO 产生。L-NAME（NOS 抑制剂）完全模拟 Ang II 对 eNOS、NO 产生和自噬水平的作用。雷帕霉素与 Ang II 合用进一步降低 NO 产生。沉默 Atg5 完全逆转 Ang II 激活的自噬水平。
我们的结果表明，Ang II 刺激通过 AT1 受体、NADPH 氧化酶、线粒体 K 通道、eNOS、Atg5 信号通路增加 HUVECs 中的自噬水平，自噬的激活有助于 Ang II 诱导的 HUVECs 功能障碍。
