doi: 10.1161/CIRCULATIONAHA.104.530832
(Circulation. 2005;111:e309.)
© 2005 American Heart Association, Inc.
Correspondence |
Letter Regarding Article by Franscini et al, "Gene Expression Profiling of Inflamed Human Endothelial Cells and Influence of Activated Protein C"
First Department of Medicine, Faculty of Clinical Medicine, University of Heidelberg, Mannheim, Germany, martina.brueckmann{at}med.ma.uni-heidelberg.de
We read with great interest the study by Franscini et al1 reporting novel antiinflammatory mechanisms of recombinant human-activated protein C (rhAPC)–dependent gene regulation in human endothelial cells. Some aspects of this work deserve further comment.
The rhAPC preparation used by the authors is registered for use in patients (Xigris, Eli Lilly) and may contain traces of bovine thrombin resulting from the manufacturing process. The addition of the direct thrombin inhibitor hirudin would have excluded any residual thrombin activity in the rhAPC preparation in experiments by Franscini et al.1 Concentrations of rhAPC used by the authors were much higher (2.5 to 20 µg/mL) than median plasma levels (
45 ng/mL) in patients. Laboratory data from the recombinant Human Activated Protein C Worldwide Evaluation in Severe Sepsis (PROWESS) trial demonstrated that a strong basis for systemic antiinflammatory effects for rhAPC is still missing.2 Thus, it is unclear whether results obtained from in vitro studies with supratherapeutic concentrations might be of clinical relevance.
Unfortunately, the authors did not mention that their observed rhAPC effects are in contrast to several previously published studies: For example, Joyce at al3 have shown that rhAPC downregulates expression of vascular adhesion molecule-1 and nuclear factor-
B in endothelial cells. Moreover, Riewald et al4 demonstrated that APC induces upregulation of the immunomodulatory monocyte chemoattractant protein-1. How do the authors interpret their data in light of these controversial results? Do they contribute this controversy to the type of endothelial cells or to the cytokine combinations that were used?
Finally, the authors conclude that rhAPC might be more effective in relatively mild systemic inflammation than in severe sepsis. Data from the PROWESS trial, however, indicate that larger absolute risk reductions were found with rhAPC in patients with higher baseline degrees of illness.5 The ADDRESS study mentioned by the authors was terminated early in February 2004 after an enrollment of 2640 sepsis patients at low risk of death because of a low likelihood of meeting the prospectively defined objective of demonstrating a reduction of 28-day mortality with rhAPC. These trial results support the concept that patients with severe stages of sepsis, especially those in a pronounced proinflammatory condition, may benefit most from rhAPC. We would like to mention that the transfer of in vitro results to in vivo conditions remains difficult, and, especially for rhAPC, further clinical investigations are needed to confirm the postulated antiinflammatory properties of this promising new drug.
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- Franscini N, Bachli EB, Blau N, Leikauf MS, Schaffner A, Schoedon G. Gene expression profiling of inflamed human endothelial cells and influence of activated protein C. Circulation. 2004; 110: 2903–2909.
[Abstract/Free Full Text]
- Dhainaut JF, Yan SB, Margolis BD, Lorente JA, Russell JA, Freebairn RC, Spapen HD, Riess H, Basson B, Johnson G III, Kinasewitz GT; PROWESS Sepsis Study Group. Drotrecogin alfa (activated) (recombinant human activated protein C) reduces host coagulopathy response in patients with severe sepsis. Thromb Haemost. 2003; 90: 642–653.[Medline]
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- Joyce DE, Gelbert L, Ciaccia A, DeHoff B, Grinnell BW. Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis. J Biol Chem. 2001; 276: 11199–11203.
[Abstract/Free Full Text]
- Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science. 2002; 296: 1880–1882.
[Abstract/Free Full Text]
- Ely EW, Laterre PF, Angus DC, Helterbrand JD, Levy H, Dhainaut JF, Vincent JL, Macias WL, Bernard GR; PROWESS Investigators. Drotrecogin alfa (activated) administration across clinically important subgroups of patients with severe sepsis. Crit Care Med. 2003; 31: 12–19.[CrossRef][Medline]
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Response
Department of Medicine, University Hospital of Zürich, Zürich, Switzerland, klinsog{at}usz.unizh.ch
We appreciate the great interest of Dr Brueckmann and colleagues in our article.1 It has been debated whether recombinant human-activated protein C (rhAPC; Xigris, Eli Lilly) might contain thrombin. In the concentrations used in our experiments, no detectable residual thrombin activity was present, and because hirudin addition had no effects on the experimental outcome at all,2 we decided to omit the addition of hirudin.
With regard to rhAPC concentrations, we considered doses equivalent to the physiological protein C concentrations of 4 µg/mL. Our dose (5 µg/mL) is in line with concentrations used by others, ranging from 8 to 180 nmol/L rhAPC (0.5 to 10 µg/mL calculated on 55 kDa molecular weight),2,3 and 2.5 to 10 µg/mL.4
We agree that our results are distinct from those published previously.2,3 In studies using human umbilical vein endothelial cells, Joyce et al performed gene expression analysis with cells preincubated with APC for 16 hours before challenge with tumor necrosis factor-
,2 and Riewald et al analyzed early-response (90 minutes) gene expression in search of the APC signaling pathway.3 The scope of our study, however, was to define genes prominently regulated in inflamed human coronary artery endothelial cells and to determine which of these genes were targets for the action of APC. We mentioned these points in the introduction and again in the discussion section of our article.
With regard to monocyte chemotactic protein-1 (MCP-1) expression, this might be regulated differently by APC. Under noninflamed conditions, APC is able to induce MCP-1.3,4 This is interesting in light of data from a murine study in which MCP-1 has been found to be protective in lethal endotoxemia.5 It is not clear, however, how much of it might be helpful or harmful in human patients. We suppose that the reduction of overexpressed MCP-1 by APC in septic conditions is part of its antiinflammatory action.
We fully agree that more refined models and further clinical studies are needed to define the antiinflammatory potential of APC.
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References |
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TopReferencesReferences |
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- Franscini N, Bachli EB, Blau N, Leikauf MS, Schaffner A, Schoedon G. Gene expression profiling of inflamed human endothelial cells and influence of activated protein C. Circulation. 2004; 110: 2903–2909.
[Abstract/Free Full Text]
- Joyce DE, Gelbert L, Ciaccia A, DeHoff B, Grinnell BW. Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis. J Biol Chem. 2001; 276: 11199–11203.
[Abstract/Free Full Text]
- Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science. 2002; 296: 1880–1882.
[Abstract/Free Full Text]
- Brueckmann M, Marx A, Weiler HM, Liebe V, Lang S, Kaden JJ, Zieger W, Borggrefe M, Huhle G, Konstantin Haase K. Stabilization of monocyte chemoattractant protein-1-mRNA by activated protein C. Thromb Haemost. 2003; 89: 149–160.[Medline]
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- Zisman DA, Kunkel SL, Strieter RM, Tsai WC, Bucknell K, Wilkowski J, Standiford TJ. MCP-1 protects mice in lethal endotoxemia. J Clin Invest. 1997; 99: 2832–2836.[Medline]
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