and endothelial dysfunction

Vascular endothelial activation is an early step during leukocyte/endothelial adhe-sion and transendothelial leukocyte mi-gration in inflammatory states. Leukocyte transmigration occurs through intercellu-lar gaps between endothelial cells. Vascu-lar endothelial cadherin (VE-cadherin) is a predominant component of endothelial adherens junctions that regulates intercel-lular gap formation. We found that tumor necrosis factor (TNF) caused tyrosine phosphorylation of VE-cadherin, separa-tion of lateral cell-cell junctions, and inter-cellular gap formation in human umbilical vein endothelial cell (HUVEC) monolay-ers. These events appear to be regulated by intracellular oxidant production through endothelial NAD(P)H (nicotin-amide adenine dinucleotide phosphate) oxidase because antioxidants and expres-sion of a transdominant inhibitor of the NADPH oxidase, p67(V204A), effectively blocked the effects of TNF on all 3 param-eters of junctional integrity. Antioxidants and p67(V204A) also decreased TNF-induced JNK activation. Dominant-nega-tive JNK abrogated VE-cadherin phos-phorylation and junctional separation, suggesting a downstream role for JNK. Finally, adenoviral delivery of the kinase dead PAK1(K298A) decreased TNF-in-duced JNK activation, VE-cadherin phos-phorylation, and lateral junctional separa-tion, consistent with the proposed involvement of PAK1 upstream of the NADPH oxidase. Thus, PAK-1 acts in con-cert with oxidase during TNF-induced oxi-dant production and loss of endothelial cell junctional integrity. (Blood. 2004;104