AMPKalpha2 deletion causes aberrant expression and activation of NAD(P)H oxidase and consequent endothelial dysfunction in vivo: role of 26S proteasomes.
AMPKalpha2 deletion causes aberrant expression and activation of NAD(P)H oxidase and consequent endothelial dysfunction in vivo: role of 26S proteasomes.
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DOI：
10.1161/circresaha.109.212530
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发表时间：
2010-04-02
影响因子：
20.1
通讯作者：
Zou MH
中科院分区：
文献类型：
--
作者：
Wang S;Zhang M;Liang B;Xu J;Xie Z;Liu C;Viollet B;Yan D;Zou MH
AMP激活蛋白激酶（AMPK）是一种能量传感器，广泛表达于血管细胞中。最近的研究表明，AMPK活化通过对抗内皮细胞中的氧化应激来改善内皮功能。AMPK如何抑制氧化应激仍有待确定。本研究旨在探讨AMPK对NAD（P）H氧化酶、氧化应激和内皮功能的调节作用。在培养的人脐静脉内皮细胞（HUVEC）和从AMPKα2缺陷小鼠分离的小鼠血管中检测AMPK活性、氧化应激标志物、NAD（P）H氧化酶亚基表达（gp 91 phox、p47 phox、p67 phox、NOX 1 -4）、NAD（P）H氧化酶介导的超氧化物生成、26 S蛋白酶体活性、IκBα降解和NF-κB核转位（p50和p65）。与野生型相比，AMPKα2−/−小鼠中乙酰胆碱（Ach）诱导的内皮依赖性舒张显著受损，同时氧化剂产生增加。此外，用超氧化物歧化酶或Tempol或夹竹桃麻素预处理主动脉显著改善AMPKα2−/−小鼠中Ach诱导的内皮依赖性舒张。对来自AMPKα2−/−小鼠的主动脉内皮细胞和表达显性阴性AMPK或AMPK α2特异性siRNA的人脐静脉内皮细胞（HUVEC）的分析显示，AMPK活性丧失增加了NAD（P）H氧化酶亚基的表达。（gp 91 phox，p47 phox，p67 phox，NOX 1 -4），NAD（P）H氧化酶介导的超氧化物生成，26 S蛋白酶体活性，IκBα降解，NF-κB（p50和p65）的核转位，而AICAR激活AMPK或过表达组成性活性AMPK则具有相反的作用。因此，我们发现LDL受体敲除（LDLr−/−）菌株中AMPKα2的基因缺失显著增加了26 S蛋白酶体活性、IκB降解、NF-κB反式激活、NAD（P）H氧化酶亚基过表达、氧化应激、内皮功能障碍和动脉粥样硬化，所有这些都在很大程度上被长期给予MG 132（一种有效的细胞渗透性蛋白酶体抑制剂）抑制。我们的结论是AMPKα2在内皮细胞中作为NAD（P）H氧化酶和ROS产生的生理抑制剂发挥作用。以这种方式，AMPK维持内皮细胞的非致动脉粥样硬化和非炎性表型。
AMP-activated protein kinase (AMPK) is an energy sensor and ubiquitously expressed in vascular cells. Recent studies suggest that AMPK activation improves endothelial function by counteracting oxidative stress in endothelial cells. How AMPK suppresses oxidative stress remains to be established. The aim of this study is to examine the effects of AMPK in regulating NAD(P)H oxidase, oxidative stress and endothelial function. AMPK activity, the markers of oxidative stress, NAD(P)H oxidase subunit expression (gp91phox, p47phox, p67phox, NOX1-4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of NF-κB (p50 and p65) were examined in cultured human umbilical vein endothelial cells (HUVEC) and mouse aortas isolated from AMPKα2 deficient mice. Compared to the wild type, acetylcholine (Ach)-induced endothelium-dependent relaxation was significantly impaired in parallel with increased production of oxidants in AMPKα2−/− mice. Further, pretreatment of aorta with either superoxide dismutase or Tempol or apocynin significantly improved Ach-induced endothelium-dependent relaxation in AMPKα2−/− mice. Analysis of aortic endothelial cells from AMPKα2−/− mice and human umbilical vein endothelial cells (HUVECs) expressing dominant negative AMPK or AMPK α2-specific siRNA revealed that loss of AMPK activity increased NAD(P)H oxidase subunit expression (gp91phox, p47phox, p67phox, NOX1-4), NAD(P)H oxidase-mediated superoxide production, 26S proteasome activity, IκBα degradation, and nuclear translocation of NF-κB (p50 and p65), whereas AMPK activation by AICAR or over-expression of constitutively active AMPK had the opposite effect. Consistently, we found that genetic deletion of AMPKα2 in LDL receptor knockout (LDLr−/−) strain markedly increased 26S proteasome activity, IκB degradation, NF-κB transactivation, NAD(P)H oxidase subunit overexpression, oxidative stress, endothelial dysfunction, and atherosclerosis, all of which were largely suppressed by chronic administration of MG132, a potent cell permeable proteasome inhibitor.. We conclude that AMPKα2 functions as a physiological suppressor of NAD(P)H oxidase and ROS production in endothelial cells. In this way AMPK maintains the non-atherogenic and non-inflammatory phenotype of endothelial cells.