Anisodamine counteracts lipopolysaccharide-induced tissue factor and plasminogen activator inhibitor-1 expression in human endothelial cells: Contribution of the NF-kappa B pathway

In this study we aimed to investigate whether the therapeutic efficacy of a nisodamine in the treatment of bacteraemic shock could - at least in part - be brought about by its direct interference with the lipopolysaccharide (L PS)-induced activation of endothelial cells. Thus, we investigated the effe ct of anisodamine on LFS-induced expression of plasminogen activator inhibi tor-1 (PAI-1) and tissue factor (TF), two major markers of endothelial acti vation. PAI-1 was measured in the conditioned media of human umbilical vein endothelial cells (HUVEC) by a specific enzyme-linked immunosorbent assay (ELISA) whereas TF activity was measured in the lysates of these cells by u sing a single step clotting assay. Results obtained in these assays were co nfirmed on the level of specific mRNA expression by Northern blotting using specific probes for human PAI-1 or TF. In order to evaluate a possible con tribution of the NF-kappaB pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HU VEC and NF-kappaB-binding oligonucleotides. When HUVEC were treated with 1 mug/ml LPS a significant increase in PAI-1 and TF activity was observed com pared with cells incubated without LPS. Anisodamine dose-dependently inhibi ted this LPS-induced upregulation of PAI-1 and TF. Anisodamine alone had no effect on the constitutive expression of PAI-1 and TF in these cells. Thes e effects were also confirmed on the level of specific PAI-1 and TF mRNA ex pression by Northern blotting. Furthermore, we could show by EMSA that anis odamine completely abolished LPS-induced NF-kappaB DNA binding activity in nuclear extracts from HUVEC treated with LPS together with anisodamine. Thu s, we provide evidence that anisodamine counteracts endothelial cell activa tion by inhibiting LPS-induced PAI-1 and TF expression in these cells. Its interference with the NF-kappaB pathway might - at least in pa rt - contrib ute to th is effect. The ability of an isodamine to counteract LPS effects on endothelial cells might be one underlying mechanism explaining its effic acy in the treatment of bacteraemic shock. Copyright (C) 2001 S. Karger AG, Basel.