A new constitutively active mutant of AMP-activated protein kinase inhibits anoxia-induced apoptosis of vascular endothelial cell
A new constitutively active mutant of AMP-activated protein kinase inhibits anoxia-induced apoptosis of vascular endothelial cell
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DOI：
10.1038/hr.2008.25
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发表时间：
2009-02-01
影响因子：
5.4
通讯作者：
Hirata, Yasunobu
中科院分区：
文献类型：
--
作者：
Nagata, Daisuke;Kiyosue, Arihiro;Hirata, Yasunobu
抑制血管内皮细胞的凋亡变化对预防血管缺氧损伤具有重要意义。amp活化蛋白激酶（AMPK）最近被发现在血管保护中起作用。虽然化学试剂5-氨基咪唑-4-羧酰胺-1- β - d -核呋喃苷（AICAR）已被用于刺激AMPK活性，但AICAR与几种非特异性反应有关。因此，我们构建了一个新的组成型活性AMPK α 1突变体（NcaAMPK），该突变体缺乏AMPK α 1的自抑制结构域，其中苏氨酸172被天冬氨酸取代。我们研究了NcaAMPK是否在缺氧条件下对血管内皮细胞具有抗凋亡作用。作为对照，NcaAMPK或绿色荧光蛋白（GFP）在人脐静脉内皮细胞（HUVECs）中过表达。HUVECs在常氧或缺氧条件下孵育40小时后，我们检测了细胞活力、caspase 3/7活性以及凋亡相关蛋白的表达和磷酸化水平。过表达ncaampk的细胞在缺氧条件下的细胞存活率提高。缺氧增加了caspase 3/7的活性，但NcaAMPK显著降低了这种活性。NcaAMPK过表达增加了蛋白激酶B/Akt Ser473和内皮型一氧化氮合酶Ser1177的磷酸化，但用一氧化氮合酶抑制剂n - g -硝基- l -精氨酸甲酯（L-NAME）预处理并没有降低过表达NcaAMPK的HUVECs的活力。此外，在缺氧条件下，共表达显性阴性Akt降低了NcaAMPK对细胞活力的改善和对poly （adp -核糖）聚合酶切割的抑制。综上所述，NcaAMPK通过激活Akt抑制缺氧诱导的血管内皮细胞凋亡，提示激活AMPK可能对缺血性血管损伤有保护作用。
The inhibition of apoptotic changes in vascular endothelial cells is important for preventing vascular damage from hypoxia. AMP-activated protein kinase (AMPK) has recently been identified as playing a role in vascular protection. Although the chemical reagent 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) has been used to stimulate AMPK activity, AICAR has been associated with several nonspecific reactions. We therefore constructed a new constitutively active mutant of AMPK alpha 1 (NcaAMPK), which lacks the autoinhibitory domain in AMPK alpha 1 and in which threonine 172 has been replaced with aspartate. We investigated whether NcaAMPK has an anti-apoptotic effect in vascular endothelial cells under anoxic conditions. NcaAMPK, or green fluorescent protein (GFP) as a control, was overexpressed in human umbilical vein endothelial cells (HUVECs). After HUVECs were incubated for 40 h under normoxic or anoxic conditions, we examined cell viability, caspase 3/7 activity, and expression and phosphorylation levels of apoptosis-related proteins. Cell viabilities under anoxic conditions were improved in NcaAMPK-overexpressing cells. Anoxia increased caspase 3/7 activity, but NcaAMPK reduced this increase significantly. NcaAMPK overexpression increased protein kinase B/Akt Ser473 and endothelial nitric oxide synthase Ser1177 phosphorylation, but pretreatment with the nitric oxide synthase inhibitor N-G-nitro-L-arginine methyl ester (L-NAME) did not decrease the viability of NcaAMPK-overexpressing HUVECs. Furthermore, co-expression of a dominant-negative Akt reduced the improvement in cell viability and the suppression of poly (ADP-ribose) polymerase cleavage by NcaAMPK under anoxic conditions. In conclusion, NcaAMPK inhibited anoxia-induced apoptosis in vascular endothelial cells through Akt activation, suggesting that activation of AMPK might protect against ischemic vascular injury.