Protection of vascular barrier integrity by activated protein C in murine models depends on protease-activated receptor-1

Summary

Protease activated receptor-1 (PAR1) mediates barrier protective signalling of activated protein C (APC) in human endothelial cells in vitro and may contribute to APC’s beneficial effects in patients with severe sepsis. Mouse models are of key importance for translational research but species differences may limit conclusions for the human system. We analysed whether mouse APC can cleave, activate and induce signalling through murine PAR1 and tested in newly established mouse models if long-term infusion of APC prevents from vascular leakage. Cell surface immunoassays demonstrated efficient cleavage of endogenous murine endothelial PAR1 by either murine or human APC. Pharmacological concentrations of APC of either species had powerful barrier protective effects on cultured murine endothelial cells that required PAR1 cleavage. Vascular endothelial growth factor-mediated hyperpermeability in the skin was reduced by either endogenously generated as well as directly infused recombinant mouse APC in wild-type mice. However APC did not significantly alter the vascular barrier function in PAR1-deficient mice. In endotoxin-challenged mice, infused APC significantly prevented from pulmonary fluid accumulation in the wild-type mice but not in mice lacking PAR1. Our results directly show that murine APC cleaves and signals through PAR1 in mouse endothelial cells. APC reduces vascular permeability in mouse models and PAR1 plays a major role in mediating these effects. Our data in vitro and in vivo support the paradigm that PAR1 contributes to protective effects of APC on vascular barrier integrity in sepsis.

Keywords

Activated protein C - endothelial cells - protease-activated receptor-1 - sepsis - vascular barrier

^* These authors contributed equally to this work.

• References

• 1 Bernard GR, Vincent JL, Laterre PF. et al. Efficacy and safety of recombinant human activated protein C for severe sepsis. N Engl J Med 2001; 344: 699-709.
• 2 Esmon CT. The protein C pathway. Chest 2003; 124: 26S-32S.
• 3 Dahlback B, Villoutreix BO. Regulation of blood coagulation by the protein C anticoagulant pathway: novel insights into structure-function relationships and molecular recognition. Arterioscler Thromb Vasc Biol 2005; 25: 1311-1320.
• 4 Warren BL, Eid A, Singer P. et al. Caring for the critically ill patient. High-dose antithrombin III in severe sepsis: a randomized controlled trial. J Am Med Assoc 2001; 286: 1869-1878.
• 5 Abraham E, Reinhart K, Opal S. et al. Efficacy and safety of tifacogin (recombinant tissue factor pathway inhibitor) in severe sepsis: a randomized controlled trial. J Am Med Assoc 2003; 290: 238-247.
• 6 Kerschen EJ, Fernandez JA, Cooley BC. et al. Endotoxemia and sepsis mortality reduction by non-anticoagulant activated protein C. J Exp Med 2007; 204: 2439-2448.
• 7 Mosnier LO, Zlokovic BV, Griffin JH. The cytoprotective protein C pathway. Blood 2007; 109: 3161-3172.
• 8 Coughlin SR. Thrombin signalling and protease-activated receptors. Nature 2000; 407: 258-264.
• 9 Riewald M, Petrovan RJ, Donner A. et al. Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 2002; 296: 1880-1882.
• 10 Guo H, Liu D, Gelbard H. et al. Activated protein C prevents neuronal apoptosis via protease activated receptors 1 and 3. Neuron 2004; 41: 563-572.
• 11 Bae JS, Yang L, Rezaie AR. Lipid raft localization regulates the cleavage specificity of protease activated receptor 1 in endothelial cells. J Thromb Haemost 2008; 6: 954-961.
• 12 Robriquet L, Collet F, Tournoys A. et al. Intravenous administration of activated protein C in Pseudo-monas-induced lung injury: impact on lung fluid balance and the inflammatory response. Respir Res 2006; 7: 41.
• 13 Abraham E. Effects of recombinant human activated protein C in human models of endotoxin administration. Proc Am Thorac Soc 2005; 2: 243-247.
• 14 Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood 2005; 105: 3178-3184.
• 15 Finigan JH, Dudek SM, Singleton PA. et al. Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation. J Biol Chem 2005; 280: 17286-17293.
• 16 McVerry BJ, Garcia JG. Endothelial cell barrier regulation by sphingosine 1-phosphate. J Cell Biochem 2004; 92: 1075-1085.
• 17 Peng X, Hassoun PM, Sammani S. et al. Protective effects of sphingosine 1-phosphate in murine endotoxin-induced inflammatory lung injury. Am J Resp Crit Care Med 2004; 169: 1245-1251.
• 18 Feistritzer C, Lenta R, Riewald M. Protease-activated receptors-1 and –2 can mediate endothelial barrier protection: role in factor Xa signaling. J Thromb Haemost 2005; 3: 2798-2805.
• 19 Feistritzer C, Schuepbach RA, Mosnier LO. et al. Protective signaling by activated protein C is mechanistically linked to protein C activation on endothelial cells. J Biol Chem 2006; 281: 20077-20084.
• 20 Schuepbach RA, Feistritzer C, Brass LF. et al. Activated protein C-cleaved protease activated receptor-1 is retained on the endothelial cell surface even in the presence of thrombin. Blood 2007; 111: 2667-2673.
• 21 Fernandez JA, Xu X, Liu D. et al. Recombinant murine-activated protein C is neuroprotective in a murine ischemic stroke model. Blood Cells Mol Dis 2003; 30: 271-276.
• 22 Li W, Zheng X, Gu J. et al. Overexpressing endothelial cell protein C receptor alters the hemostatic balance and protects mice from endotoxin. J Thromb Haemost 2005; 3: 1351-1359.
• 23 Edgell CJ, McDonald CC, Graham JB. Permanent cell line expressing human factor VIII-related antigen established by hybridization. Proc Natl Acad Sci USA 1983; 80: 3734-3737.
• 24 Riewald M, Ruf W. Protease-activated receptor-1 signaling by activated protein C in cytokine-perturbed endothelial cells is distinct from thrombin signaling. J Biol Chem 2005; 280: 19808-19814.
• 25 Cantwell AM, Di Cera E. Rational design of a potent anticoagulant thrombin. J Biol Chem 2000; 275: 39827-39830.
• 26 Gruber A, Fernandez JA, Bush L. et al. Limited generation of activated protein C during infusion of the protein C activator thrombin analog W215A/E217A in primates. J Thromb Haemost 2006; 4: 392-397.
• 27 Cheng T, Liu D, Griffin JH. et al. Activated protein C blocks p53-mediated apoptosis in ischemic human brain endothelium and is neuroprotective. Nat Med 2003; 9: 338-342.
• 28 McVerry BJ, Garcia JG. In vitro and in vivo modulation of vascular barrier integrity by sphingosine 1-phosphate: mechanistic insights. Cell Signal 2005; 17: 131-139.
• 29 Guha M, Mackman N. LPS induction of gene expression in human monocytes. Cell Signal 2001; 13: 85-94.
• 30 Weis SM, Cheresh DA. Pathophysiological consequences of VEGF-induced vascular permeability. Nature 2005; 437: 497-504.
• 31 Coughlin SR, Camerer E. PARticipation in inflammation. J Clin Invest 2003; 111: 25-27.
• 32 Cunningham MA, Rondeau E, Chen X. et al. Protease-activated receptor 1 mediates thrombin-dependent, cell- mediated renal inflammation in crescentic glomerulonephritis. J Exp Med 2000; 191: 455-462.
• 33 Sevastos J, Kennedy SE, Davis DR. et al. Tissue factor deficiency and PAR-1 deficiency are protective against renal ischemia reperfusion injury. Blood 2007; 109: 577-583.
• 34 Vergnolle N, Cellars L, Mencarelli A. et al. A role for proteinase-activated receptor-1 in inflammatory bowel diseases. J Clin Invest 2004; 114: 1444-1456.
• 35 Pawlinski R, Pedersen B, Schabbauer G. et al. Role of tissue factor and protease-activated receptors in a mouse model of endotoxemia. Blood 2004; 103: 1342-1347.
• 36 Camerer E, Cornelissen I, Kataoka H. et al. Roles of protease-activated receptors in a mouse model of endotoxemia. Blood 2006; 107: 3912-3921.
• 37 Kaneider NC, Leger AJ, Agarwal A. et al. ‘Role reversal’ for the receptor PAR1 in sepsis-induced vascular damage. Nat Immunol 2007; 8: 1303-1312.