Akt activates NOS3 and separately restores barrier integrity in H2O2-stressed human cardiac microvascular endothelium
Akt activates NOS3 and separately restores barrier integrity in H2O2-stressed human cardiac microvascular endothelium
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DOI：
10.1152/ajpheart.00501.2008
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发表时间：
2008-12-01
影响因子：
4.8
通讯作者：
Hoek, Terry L. Vanden
中科院分区：
文献类型：
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作者：
Dossumbekova, Anar;Berdyshev, Evgeny V.;Hoek, Terry L. Vanden
Dossumbekova A，Berdyshev EV，Gorshkova I，Shao Z，Li C，Long P，Joshi A，Natarajan V，Vanden Hoek TL。 Akt 激活 NOS3 并分别恢复 H2O2 应激的人心脏微血管内皮屏障完整性。 Am J Physiol Heart Circ Physiol 295：H2417 -H2426，2008。首次发表于 2008 年 10 月 17 日； doi：10.1152/ajpheart。 00501.2008。 -微血管内皮的完整性是心肌收缩力的重要调节因子。心脏骤停复苏后几分钟内观察到的活性氧（ROS）应激增加可能会改变微血管屏障的完整性。 Akt 及其下游靶标一氧化氮 (NO) 合酶 (NOS) 3 可以在 ROS 应激期间保护屏障完整性，但很少有工作研究人心脏微血管内皮细胞 (HCMVEC) 中的这些氧化应激反应。因此，我们研究了 HCMVEC 中 ROS 如何通过 Akt 及其下游靶标 NOS3 影响屏障功能和 NO 生成。暴露于 500 μM H2O2 的 HCMVEC 在 10 分钟内增加了 Ser-473 和 Thr-308 位点的 Akt 磷酸化，这一效应被磷脂酰肌醇 3-激酶抑制剂 LY-294002 阻断。 H2O2 还诱导与 NOS3 Ser-1177 位点磷酸化和 Thr-495 去磷酸化相关的 NO 生成，LY294002 和 Akt 抑制剂 Akt/PKB 信号抑制剂-2 (API-2) 可减弱 Ser-1177 的作用。 H2O2 在几分钟内引起 HCMVEC 屏障的显着破坏，但在 30 分钟内开始恢复，并在数小时内恢复正常。 NOS抑制剂N-omega-硝基-L-精氨酸甲酯（200μM）可阻断NO生成，但对H2O2诱导的屏障通透性或屏障完整性的恢复没有影响。相比之下，Akt 抑制剂 API-2 消除了 HCMVEC 屏障恢复。这些结果表明 HCMVEC 中的氧化应激通过 Akt 激活 NOS3。 NOS3/NO 不参与 H2O2 影响的 HCMVEC 屏障功能的调节。独立于 NOS3 调节，Akt 被证明对于恢复 HCMVEC 屏障完整性至关重要。
Dossumbekova A, Berdyshev EV, Gorshkova I, Shao Z, Li C, Long P, Joshi A, Natarajan V, Vanden Hoek TL. Akt activates NOS3 and separately restores barrier integrity in H2O2-stressed human cardiac microvascular endothelium. Am J Physiol Heart Circ Physiol 295: H2417 -H2426, 2008. First published October 17, 2008; doi: 10.1152/ajpheart. 00501.2008. -The integrity of microvascular endothelium is an important regulator of myocardial contractility. Microvascular barrier integrity could be altered by increased reactive oxygen species (ROS) stress seen within minutes after cardiac arrest resuscitation. Akt and its downstream target nitric oxide (NO) synthase (NOS) 3 can protect barrier integrity during ROS stress, but little work has studied these oxidant stress responses in human cardiac microvascular endothelial cells (HCMVEC). We, therefore, studied how ROS affects barrier function and NO generation via Akt and its downstream target NOS3 in HCMVEC. HCMVEC exposed to 500 mu M H2O2 had increased Akt phosphorylation within 10 min at both Ser-473 and Thr-308 sites, an effect blocked by the phosphatidylinositol 3-kinase inhibitor LY-294002. H2O2 also induced NO generation that was associated with NOS3 Ser-1177 site phosphorylation and Thr-495 dephosphorylation, with Ser-1177 effects attenuated by LY294002 and an Akt inhibitor, Akt/PKB signaling inhibitor-2 (API-2). H2O2 induced significant barrier disruption in HCMVEC within minutes, but recovery started within 30 min and normalized over hours. The NOS inhibitor N-omega-nitro-L-arginine methyl ester (200 mu M) blocked NO generation but had no effect on H2O2-induced barrier permeability or the recovery of barrier integrity. By contrast, the Akt inhibitor API-2 abrogated HCMVEC barrier restoration. These results suggest that oxidant stress in HCMVEC activates NOS3 via Akt. NOS3/NO are not involved in the regulation of H2O2-affected barrier function in HCMVEC. Independent of NOS3 regulation, Akt proves to be critical for the restoration of barrier integrity in HCMVEC.