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Recombinant human activated protein C upregulates cyclooxygenase-2 and release of prostaglandins from cultured human endothelial cells via the endothelial protein C receptor
Critical Care volume 8, Article number: P109 (2004)
Cyclooxygenase-2 (COX-2) is the inducible COX isoform that catalyzes the formation of prostaglandins in response to proinflammatory cytokines. Prostacyclin, the major prostanoid generated by endothelial cells, is a potent inhibitor of platelet aggregation and a powerful vasodilator. Although the role of Drotrecogin alfa (activated) (recombinant human activated protein C [rhAPC]) in modulating microvascular coagulation through the inhibition of thrombin generation has been well studied in experimental and clinical settings of severe sepsis, little is known about its effects on prostanoid release from endothelial cells.
The effect of rhAPC (1–20 μg/ml) on the expression of COX-2-mRNA in human umbilical vein endothelial cells (HUVEC) was measured by a colorimetric assay (Quantikine assay™). The COX-2 protein content in endothelial cells was determined by western blots. Cell supernantants were assayed for 6-keto-PFG1α, the stable hydrolysis product of prostacyclin, by ELISA. Statistical analysis was performed by unpaired Student's t test and ANOVA.
rhAPC dose-dependently upregulated COX-2 mRNA in endothelial cells after an incubation time of 4 hours (controls, 11.7 ± 0.6 amol/ml; HUVEC treated with 20 μg/ml rhAPC, 18.4 ± 0.8 amol/ml; mean ± SEM, P < 0.001). Western blot analysis revealed an increase of COX-2 protein content after a 10-hour treatment with rhAPC (1 μg/ml). rhAPC dose-dependently increased 6-keto-PFG1α -levels in HUVEC supernatants (controls, 62.5 ± 8.7 pg/ml; HUVEC treated with 20 μg/ml rhAPC, 151.7 ± 16.3 pg/ml, P < 0.001). As shown by antagonists to the thrombin receptor PAR-1 (WEDE 15 and ATAP2) and by a monoclonal antibody (RCR-252) against the endothelial protein C receptor (EPCR), the effect of rhAPC on COX-2 mRNA upregulation was mediated by the EPCR receptor.
The ability of rhAPC to upregulate COX-2 in human endothelial cells, as well as the release of prostanoids, very probably mediated by the EPCR receptor, may represent a new molecular mechanism, by which oxygen delivery at the site of injury may be improved and intravascular aggregation of platelets may be reduced, thereby contributing to the efficacy of rhAPC in systemic inflammation and sepsis.
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Brueckmann, M., Fukudome, K., Horn, S. et al. Recombinant human activated protein C upregulates cyclooxygenase-2 and release of prostaglandins from cultured human endothelial cells via the endothelial protein C receptor. Crit Care 8 (Suppl 1), P109 (2004). https://doi.org/10.1186/cc2576