Role of NF-κB activation in LPS-induced endothelial barrier breakdown.
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.
Department of Surgery I, University of Würzburg, Oberdürrbacher Str. 6, 97080 Würzburg, Germany. Schlegel_N@chirurgie.uni-wuerzburg.de
Nicolas
N
Schlegel
Rhea
R
Leweke
Michael
M
Meir
Christoph-Thomas
CT
Germer
Jens
J
Waschke
D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D004727:Endothelium / Q000187:drug effects / Q000473:pathology; D004789:Enzyme Activation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D006651:Histocytochemistry; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D013552:Swine; D000097782:NF-kappaB-Inducing Kinase
D008070:Lipopolysaccharides; D017346:Protein Serine-Threonine Kinases; D000097782:NF-kappaB-Inducing Kinase