Department of Cardiology, Nanjing First Hospital, Nanjing Medical University, 210006, Nanjing, China.
Department of Cardiology, Guangzhou Red Cross Hospital, Jinan University, 510020, Guangzhou, China.
Biochim Biophys Acta Mol Cell Res. 2018 May;1865(5):709-720. doi: 10.1016/j.bbamcr.2018.02.005. Epub 2018 Feb 18.
Uncoupled endothelial nitric oxide synthase (eNOS) produces O instead of nitric oxide (NO). Earlier, we reported rapamycin, an autophagy inducer and inhibitor of cellular proliferation, attenuated low shear stress (SS) induced O production. Nevertheless, it is unclear whether autophagy plays a critical role in the regulation of eNOS uncoupling. Therefore, this study aimed to investigate the modulation of autophagy on eNOS uncoupling induced by low SS exposure. We found that low SS induced endothelial O burst, which was accompanied by reduced NO release. Furthermore, inhibition of eNOS by L-NAME conspicuously attenuated low SS-induced O releasing, indicating eNOS uncoupling. Autophagy markers such as LC3 II/I ratio, amount of Beclin1, as well as ULK1/Atg1 were increased during low SS exposure, whereas autophagic degradation of p62/SQSTM1 was markedly reduced, implying impaired autophagic flux. Interestingly, low SS-induced NO reduction could be reversed by rapamycin, WYE-354 or ATG5 overexpression vector via restoration of autophagic flux, but not by N-acetylcysteine or apocynin. eNOS uncoupling might be ascribed to autophagic flux blockade because phosphorylation of eNOS Thr495 by low SS or PMA stimulation was also regulated by autophagy. In contrast, eNOS acetylation was not found to be regulated by low SS and autophagy. Notably, although low SS had no influence on eNOS Ser1177 phosphorylation, whereas boosted eNOS Ser1177 phosphorylation by rapamycin were in favor of the eNOS recoupling through restoration of autophagic flux. Taken together, we reported a novel mechanism for regulation of eNOS uncoupling by low SS via autophagy-mediated eNOS phosphorylation, which is implicated in geometrical nature of atherogenesis.
解偶联内皮型一氧化氮合酶（eNOS）产生 O 而不是一氧化氮（NO）。我们之前曾报道，雷帕霉素是一种自噬诱导剂和细胞增殖抑制剂，可减弱低切应力（SS）诱导的 O 产生。然而，尚不清楚自噬是否在调节 eNOS 解偶联中发挥关键作用。因此，本研究旨在探讨自噬在低 SS 暴露诱导的 eNOS 解偶联中的调节作用。我们发现，低 SS 诱导内皮细胞 O 爆发，同时伴随着 NO 释放减少。此外，L-NAME 抑制 eNOS 明显减弱了低 SS 诱导的 O 释放，表明 eNOS 解偶联。在低 SS 暴露期间，自噬标记物如 LC3 II/I 比值、Beclin1 量以及 ULK1/Atg1 增加，而 p62/SQSTM1 的自噬降解明显减少，表明自噬流受损。有趣的是，雷帕霉素、WYE-354 或 ATG5 过表达载体通过恢复自噬流可以逆转低 SS 诱导的 NO 减少，而 N-乙酰半胱氨酸或 apocynin 则不能。eNOS 解偶联可能归因于自噬流阻断，因为低 SS 或 PMA 刺激引起的 eNOS Thr495 磷酸化也受到自噬的调节。相反，eNOS 乙酰化不受低 SS 和自噬调节。值得注意的是，虽然低 SS 对 eNOS Ser1177 磷酸化没有影响，但雷帕霉素促进的 eNOS Ser1177 磷酸化有利于通过恢复自噬流使 eNOS 重新偶联。总之，我们报道了低 SS 通过自噬介导的 eNOS 磷酸化调节 eNOS 解偶联的新机制，这与动脉粥样硬化的几何性质有关。
