Department of Anesthesiology, University of Alabama, Birmingham, Alabama, USA.
FASEB J. 2011 Oct;25(10):3356-65. doi: 10.1096/fj.10-179218. Epub 2011 Jun 16.
The vascular endothelium responds to damage through activation of multiple signaling events that restore cell-cell adhesion and vascular integrity. However, the molecular mechanisms that integrate these events are not clearly defined. Herein, we identify a previously unexpected role for adenosine monophosphate-activated protein kinase (AMPK) in pulmonary microvascular endothelial cell (PMVEC) repair. PMVECs selectively express the AMPKα1 catalytic subunit, pharmacological and short hairpin RNA-mediated inhibition of which attenuates Ca(2+) entry in these cells induced by the inflammatory Ca(2+)-signaling mimetic thapsigargin. We find that AMPKα1 activity is required for the formation of PMVEC cell-cell networks in a prorepair environment and for monolayer resealing after wounding. Decreasing AMPKα1 expression reduces barrier resistance in PMVEC monolayers, results consistent with a role for AMPKα1 in cell-cell adhesion. AMPKα1 colocalizes and coimmunoprecipitates with the adherens junction protein N-cadherin and cofractionates with proteins selectively expressed in caveolar membranes. Assessment of permeability, by measuring the filtration coefficient (K(f)) in isolated perfused lungs, confirmed that AMPK activation contributes to barrier repair in vivo. Our findings thus provide novel evidence for AMPKα1 in Ca(2+) influx-mediated signaling and wound repair in the endothelium.
血管内皮细胞通过激活多种信号事件来响应损伤，这些信号事件恢复细胞间的黏附和血管完整性。然而，整合这些事件的分子机制尚不清楚。在此，我们发现了一个意想不到的作用，即一磷酸腺苷激活的蛋白激酶（AMPK）在肺微血管内皮细胞（PMVEC）修复中的作用。PMVEC 选择性表达 AMPKα1 催化亚基，其药理学和短发夹 RNA 介导的抑制作用可减弱炎症性 Ca(2+)信号模拟物 thapsigargin 诱导的这些细胞中的 Ca(2+)内流。我们发现，在促进修复的环境中，AMPKα1 活性对于 PMVEC 细胞间网络的形成以及在损伤后单层细胞的重新封闭是必需的。降低 AMPKα1 表达会降低 PMVEC 单层的屏障阻力，这与 AMPKα1 在细胞间黏附中的作用一致。AMPKα1 与黏着连接蛋白 N-钙黏蛋白共定位和共免疫沉淀，并与选择性表达在小窝膜中的蛋白质共分馏。通过测量在分离灌注的肺中的过滤系数（K(f)）来评估通透性，证实了 AMPK 激活有助于体内屏障修复。因此，我们的研究结果为 AMPKα1 在钙内流介导的信号和内皮细胞伤口修复中的作用提供了新的证据。
