Role of NF-κB activation in LPS-induced endothelial barrier breakdown
Role of NF-κB activation in LPS-induced endothelial barrier breakdown
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DOI：
10.1007/s00418-012-0983-7
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发表时间：
2012-10-01
影响因子：
2.3
通讯作者：
Waschke, Jens
中科院分区：
文献类型：
--
作者：
Schlegel, Nicolas;Leweke, Rhea;Waschke, Jens
脓毒症时血管内皮细胞屏障的破坏导致器官衰竭。有效的脓毒症诱导剂脂多糖(LPS)破坏内皮细胞屏障的关键机制存在争议。在这里，我们测试了一种假设，即核因子-kappaB的激活在内皮屏障的破坏中起关键作用。在猪肺动脉内皮细胞(PAEC)和人真皮微血管内皮细胞(HDMEC)的单层细胞中应用脂多糖，核转录因子-kappaB的亚基p65移位到细胞核内，免疫染色显示核转录因子-kappa B的快速和持续的激活。内皮细胞跨内皮细胞电阻(TER)和细胞间隙形成的测定显示，内毒素处理3h后，内皮细胞屏障特性明显破坏。有趣的是，单层在10h后开始自发恢复。cAMP的增加阻止了内毒素诱导的内皮屏障特性的丧失，但不能阻止核因子-kappaB的激活。应用细胞可穿透性Nemo结合结构域(NBD)合成肽可以有效地阻止核因子-kappaB的激活，但不能阻止内毒素诱导的TER丢失和细胞间隙的形成。NBD多肽单独应用不会改变内皮屏障的特性，但当与脂多糖联合应用时，会增强屏障的破坏作用。同样，siRNA介导的敲除HDMECs中p65的基因并不能阻止内毒素诱导的屏障破坏。已知的核因子-kappa B衍生蛋白的靶点小窝蛋白或血管扩张剂刺激的磷蛋白(VASP)的表达仍然不会因内皮细胞的内毒素处理而改变。综上所述，我们的数据表明，由内毒素激活的核因子-kappaB并不是破坏内皮细胞屏障特性的关键因素。相反，我们的数据表明，核因子-kappa B的激活是救援机制的一部分。
Endothelial barrier breakdown contributes to organ failure in sepsis. The key mechanism by which the potent sepsis inductor lipopolysaccharide (LPS) disrupts the endothelial barrier is controversial. Here, we tested the hypothesis that NF-kappa B activation is critically involved in endothelial barrier breakdown. Application of LPS to monolayers of porcine pulmonary artery endothelial cells (PAEC) and human dermal microvascular endothelial cells (HDMEC) induced a rapid and sustained activation of NF-kappa B as revealed by translocation of its subunit p65 into the nuclei in nuclear extraction assays and by immunostaining. Measurements of transendothelial electrical resistance (TER) and intercellular gap formation demonstrated significant breakdown of endothelial barrier properties following LPS treatment for 3 h. Interestingly, monolayers recovered spontaneously beginning after 10 h. Increased cAMP prevented LPS-induced loss of endothelial barrier properties, but did not block NF-kappa B activation. Application of the cell-permeable NEMO-binding domain (NBD) synthetic peptide was effective to prevent NF-kappa B activation, but did neither block LPS-induced loss of TER nor intercellular gap formation. NBD peptide alone did not alter endothelial barrier properties, but enhanced the barrier-compromising effects when applied in combination with LPS. Similarly, siRNA-mediated knock-down of p65 in HDMECs did not prevent LPS-induced barrier breakdown. Known targets of NF-kappa B-derived protein expression of caveolin or vasodilator-stimulated phosphoprotein (VASP) remained unaltered by LPS treatment of endothelial cells. In summary, our data indicate that NF-kappa B activation by LPS is not critically involved in disruption of endothelial barrier properties. Rather, our data suggest that NF-kappa B activation acts as a part of a rescue mechanism.