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Drotregocin alfa (activated) inhibits degradation of cytokine-mRNA in an endothelial model of inflammation

Background

The activated protein C (APC) pathway has been suggested to be a common link between coagulation and inflammation. APC may function to restore hemostasis via modulation of cytokine expression. We investigated the effect of Drotrecogin alfa (activated) (recombinant human activated protein C [rhAPC]) on the expression of monocyte chemoattractant protein-1 (MCP-1), interleukin-6 (IL-6) and IL-8 in human umbilical vein endothelial cells (HUVEC) in the presence and absence of tumor necrosis factor-alpha (TNF-alpha). MCP-1, IL-6 and IL-8 are mediators of inflammation and their gene expression is controlled by the activation of the transcription factor nuclear factor-kappa B (NF-κB).

Results

rhAPC (2.5–20 μg/ml) upregulated the amount of MCP-1-mRNA and IL-8-mRNA and caused a time-dependent and dose-dependent increase in MCP-1-, IL-6- and IL-8-protein production (P < 0.001 for rhAPC 5 μg/ml at 4–24 hours) in HUVEC. Experiments were conducted to evaluate the effect of rhAPC on mRNA degradation and mRNA stability independently of its possible effects on gene transcription. After stimulation of mRNA transcription by TNF-alpha (0.1–1 ng/ml) for 3 hours, HUVEC were treated with actinomycin D (1 μg/ml), preventing new synthesis of transcript, in the presence or absence of rhAPC. HUVEC receiving rhAPC contained more MCP-1-mRNA and IL-8-mRNA after 1 hour and up to 8 hours than controls, suggesting an inhibitory effect of rhAPC on mRNA degradation. Electrophoretic mobility shift assays (EMSA) revealed that APC attenuated NF-κB activity implying that NF-κB may not be involved in the upregulatory effect of rhAPC on MCP-1, IL-6 and IL-8 production.

Conclusions

The ability of APC to upregulate the production of MCP-1, IL-6 and IL-8, most likely by increasing the stability of MCP-1-mRNA rather than by transcriptional activation via NF-κB, identifies a novel post-transcriptional pathway, by which APC may control the local inflammatory reaction, thereby modulating the extent of endothelial injury.

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Brueckmann, M., Weiler, H., Liebe, V. et al. Drotregocin alfa (activated) inhibits degradation of cytokine-mRNA in an endothelial model of inflammation. Crit Care 7, P019 (2003). https://doi.org/10.1186/cc1908

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  • DOI: https://doi.org/10.1186/cc1908

Keywords

  • Human Umbilical Vein Endothelial Cell
  • Electrophoretic Mobility Shift Assay
  • Actinomycin
  • mRNA Stability
  • Monocyte Chemoattractant