Abstract
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-κB 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-κB 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-κB activation. Application of the cell-permeable NEMO-binding domain (NBD) synthetic peptide was effective to prevent NF-κB 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-κB-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-κB activation by LPS is not critically involved in disruption of endothelial barrier properties. Rather, our data suggest that NF-κB activation acts as a part of a rescue mechanism.
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Acknowledgments
We are grateful to Veronika Heimbach, Alexia Witchen and Nadja Niedermeier for skilful technical assistance. These studies were supported by a grant from the Interdisziplinäre Zentrum für Klinische Forschung (IZKF) Z3/2 (N.S.), A102 (N.S. and J.W.) and SFB 688 (J.W.) and DFG 1962/2-1 (N.S.). Grant: These studies were supported by a grant from the Interdisziplinäre Zentrum für Klinische Forschung (IZKF) Z3/2 (N.S.), A102 (N.S. and J.W.), SFB 688 (J.W.), DFG 1962/2-1.
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N. Schlegel and R. Leweke contributed equally to this work.
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Schlegel, N., Leweke, R., Meir, M. et al. Role of NF-κB activation in LPS-induced endothelial barrier breakdown. Histochem Cell Biol 138, 627–641 (2012). https://doi.org/10.1007/s00418-012-0983-7
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DOI: https://doi.org/10.1007/s00418-012-0983-7