title: Anisodamine counteracts lipopolysaccharide-induced tissue factor and plasminogen activator inhibitor-1 expression in human endothelial cells: contribution of the NF-kappa b pathway.
abstract: In this study we aimed to investigate whether the therapeutic efficacy of anisodamine in the treatment of bacteraemic shock could--at least in part--be brought about by its direct interference with the lipopolysaccharide (LPS)-induced activation of endothelial cells. Thus, we investigated the effect of anisodamine on LPS-induced expression of plasminogen activator inhibitor-1 (PAI-1) and tissue factor (TF), two major markers of endothelial activation. 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 using a single step clotting assay. Results obtained in these assays were confirmed 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 contribution of the NF-kappa B pathway on the effects observed, electrophoretic mobility shift assays (EMSA) were performed using nuclear extracts from HUVEC and NF-kappa B-binding oligonucleotides. When HUVEC were treated with 1 microg/ml LPS a significant increase in PAI-1 and TF activity was observed compared with cells incubated without LPS. Anisodamine dose-dependently inhibited 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. These effects were also confirmed on the level of specific PAI-1 and TF mRNA expression by Northern blotting. Furthermore, we could show by EMSA that anisodamine completely abolished LPS-induced NF-kappa B DNA binding activity in nuclear extracts from HUVEC treated with LPS together with anisodamine. Thus, we provide evidence that anisodamine counteracts endothelial cell activation by inhibiting LPS-induced PAI-1 and TF expression in these cells. Its interference with the NF-kappa B pathway might - at least in part - contribute to this effect. The ability of anisodamine to counteract LPS effects on endothelial cells might be one underlying mechanism explaining its efficacy in the treatment of bacteraemic shock.
authors.affiliation: Department of Vascular Biology and Thrombosis Research, University of Vienna, Austria.
authors.forename: Q R
authors.initials: QR
authors.lastname: Ruan
authors.forename: W J
authors.initials: WJ
authors.lastname: Zhang
authors.forename: P
authors.initials: P
authors.lastname: Hufnagl
authors.forename: C
authors.initials: C
authors.lastname: Kaun
authors.forename: B R
authors.initials: BR
authors.lastname: Binder
authors.forename: J
authors.initials: J
authors.lastname: Wojta
mesh_terms: D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology* / Q000627:therapeutic use; D002478:Cells, Cultured / Q000187:drug effects; D004247:DNA / Q000378:metabolism; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects*; D006801:Humans; D008070:Lipopolysaccharides / Q000037:antagonists & inhibitors* / Q000633:toxicity; D016328:NF-kappa B / Q000502:physiology*; D017395:Plasminogen Activator Inhibitor 1 / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D011485:Protein Binding / Q000187:drug effects; D012772:Shock, Septic / Q000188:drug therapy / Q000503:physiopathology; D012991:Solanaceous Alkaloids / Q000494:pharmacology* / Q000627:therapeutic use; D013925:Thromboplastin / Q000096:biosynthesis* / Q000235:genetics; D010959:Tissue Plasminogen Activator / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*; D014471:Umbilical Veins
chemical_list: D000894:Anti-Inflammatory Agents, Non-Steroidal; D008070:Lipopolysaccharides; D016328:NF-kappa B; D017395:Plasminogen Activator Inhibitor 1; D012991:Solanaceous Alkaloids; C003922:anisodamine; D004247:DNA; D013925:Thromboplastin; D010959:Tissue Plasminogen Activator