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 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.

[Monocyte chemotactic factor produced by human umbilical vein endothelial cells in vitro].

The blood monocytes have been well known as one of the origins of foam cells in atherosclerotic plaque, but the mechanism controlling the migration of monocytes into the subendothelial space is still unclear. The chemotactic activity of the conditioned medium from cultured human umbilical vein endothelial cells for human blood monocytes was investigated by micropore filter assay; meanwhile, heat stability and enzyme digestion assays were undertaken. The results showed that the conditioned medium from the cultured endothelial cells was significantly chemotactic rather than chemokinetic for monocytes. The conditioned medium from the cultured endothelial cells still retained the chemotactic activity for monocytes although it was heated at 80C and 100C for 15 min respectively, whereas after digestion with protease, the chemotactic activity vanished. From the above-mentioned facts, it is reasonable to believe that cultured human umbilical vein endothelial cells can secrete a chemotactic factor for monocytes, which is a kind of peptide.