Recombinant human activated protein C upregulates cyclooxygenase-2 expression in endothelial cells via binding to endothelial cell protein C receptor and activation of protease-activated receptor-1

Martina Brueckmann; Sarah Horn; Siegfried Lang; Kenji Fukudome; Adriane Schulze Nahrup; Ursula Hoffmann; Jens J. Kaden; Martin Borggrefe; Karl K. Haase; Guenter Huhle

doi:10.1160/TH04-08-0511

Thrombosis and Haemostasis, Inhaltsverzeichnis

Thromb Haemost 2005; 93(04): 743-750
DOI: 10.1160/TH04-08-0511

Endothelium and Vascular Development

Schattauer GmbH

Recombinant human activated protein C upregulates cyclooxygenase-2 expression in endothelial cells via binding to endothelial cell protein C receptor and activation of protease-activated receptor-1

Martina Brueckmann

¹1^st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany

,

Sarah Horn

¹1^st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany

,

Siegfried Lang

¹1^st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany

,

Kenji Fukudome

²Department of Immunology, Saga Medical School, Saga, Japan

,

Adriane Schulze Nahrup

¹1^st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany

,

Ursula Hoffmann

¹1^st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany

,

Jens J. Kaden

¹1^st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany

,

Martin Borggrefe

¹1^st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany

,

Karl K. Haase

¹1^st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany

,

Guenter Huhle^*

¹1^st Department of Medicine, Faculty of Clinical Medicine Mannheim, University of Heidelberg, Mannheim, Germany

› Institutsangaben

Abstract

Summary

Prostacyclin (PGI₂) has beneficial cytoprotective properties, is a potent inhibitor of platelet aggregation and has been reported to improve microcirculatory blood flow during sepsis. The formation of PGI₂ in response to proinflammatory cytokines is catalysed by the inducible cyclooxygenase (COX) isoform COX-2. Recombinant human activated protein C (rhAPC, drotrecogin alfa (activated)) was shown to have multiple biological activities in vitro and to promote resolution of organ dysfunction in septic patients. Whether rhAPC exerts its beneficial effects by modulating prostanoid generation is unknown up to now. It was therefore the aim of the study to examine the in vitro effect of rhAPC on COX-2-mRNA-expression and PGI₂ release from human umbilical vein endothelial cells (HUVEC). We found that rhAPC, at supra-therapeutical concentrations (500ng/ml-20μg/ ml), upregulated the amount of COX-2-mRNA in HUVEC at t=3–9h and caused a time- and dose-dependent release of 6-keto PGF_1α, the stable hydrolysis product of prostacyclin. RhAPC further increased the stimulating effect of tumor necrosis factor-α (TNF-α) and thrombin on COX-2-mRNA-levels. Transcript levels of cyclooxygenase-1 (COX-1) and prostagland-in I2 synthase, however, were unaffected by the stimulation with rhAPC or thrombin. The upregulatory effect on COX2-mRNA levels was specific for rhAPC since the zymogen protein C in equimolar concentrations had no effect on COX-2-mRNA-levels or 6keto PGF_1α-release. Western Blot analysis revealed an increase of COX-2-protein content in HUVEC after treatment with rhAPC. As shown by experiments using monoclonal antibodies against the thrombin receptor PAR-1 (mAb=ATAP2) and against the endothelial protein C receptor (EPCR; mAb=RCR-252), the effect of rhAPC on COX-2-mRNA up-regulation was mediated by binding to the EPCR-receptor and signaling via PAR-1. These results demonstrate that induction of COX-2-expression is an important response of HUVEC to stimulation with rhAPC and may represent a new molecular mechanism, by which rhAPC promotes upregulation of prostanoid production in human endothelium.

Keywords

Activated protein C - drotrecogin alfa (activated) - cyclooxygenase-2 - prostacyclin - endothelium

Volltext

Referenzen

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