Activated protein C downregulates p38 mitogen-activated protein kinase and improves clinical parameters in an in-vivo model of septic shock

Summary

Despite the success of the anti-coagulant protease protein C (PC) in treating septic shock in humans, the signaling pathways used are still unclear. To explore the effects of treatment with PC zymogen and its activated form aPC in a setting of sepsis, we employed a piglet model of endotoxic shock. In the aPC group, we observed a 65%-90% reduction in plasmaTNF-alpha levels and a concomitant clinical improvement. Unexpectedly, administration of aPC also resulted in stabilization of the plasma pH above 7.2. Moreover, phosphorylated p38 mitogen-activated protein kinase (p38MAPK) was virtually absent in the livers of those piglets receiving aPC. In cultured human umbilical vein endothelial cells, we observed that nanomolar concentrations of PC and aPC inhibited the phosphorylation of p38MAPK. Furthermore, we showed that the regulation of the pro-apoptotic cell cycle regulator p53 by PC and aPC is dependent on the reduction of p38MAPK activation. The transduction of these effects involves all three receptors associated with protein C signaling, namely endothelial protein C receptor, protease-activated receptor 1, and sphingosine 1-phosphate receptor 1. Ultimately, this study elucidates novel signaling pathways regulated by protein C and emphasises the pivotal importance of its multiple modes of action beyond anticoagulation. APC’s clinical success may, in part, be due to p38MAPK inhibition.

Keywords

Protein C - sepsis - signal transduction - p38MAPK - apoptosis

• References

• 1 Hotchkiss RS, Karl IE. The pathophysiology and treatment of sepsis. N Engl J Med 2003; 348: 138-150.
• 2 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.
• 3 Joyce DE, Gelbert L, Ciaccia A. et al. Gene expression profile of antithrombotic protein C defines new mechanisms modulating inflammation and apoptosis. J Biol Chem 2001; 276: 11199-11203.
• 4 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.
• 5 Isobe H, Okajima K, Uchiba M. et al. Activated protein C prevents endotoxin-induced hypotension in rats by inhibiting excessive production of NO. Circulation 2001; 104: 1171-1175.
• 6 Mizutani A, Okajima K, Uchiba M. et al. Activated protein C reduces ischemia/ reperfusion-induced renal injury in rats by inhibiting leukocyte activation. Blood 2000; 95: 3781-3787.
• 7 Petter-Puchner AH, Sieber J, Hopf R. et al. NONactivated protein C as post-treatment after spinal cord compression injury in rats. Acta Neurochir (Wien) 2006; 148: 765-771.
• 8 Castellino FJ, Ganopolsky JG, Noria F. et al. Focal arterial inflammation is augmented in mice with a deficiency of the protein C gene. Thromb Haemost 2006; 96: 794-801.
• 9 Dinarello CA. Proinflammatory and anti-inflammatory cytokines as mediators in the pathogenesis of septic shock. Chest 1997; 112: 321S-329S.
• 10 White B, Schmidt M, Murphy C. et al. Activated protein C inhibits lipopolysaccharide-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) and tumour necrosis factor alpha (TNF-alpha) production in the THP-1 monocytic cell line. Br J Haematol 2000; 110: 130-134.
• 11 Brueckmann M, Hoffmann U, Dvortsak E. et al. Drotrecogin alfa (activated) inhibits NF-kappa B activation and MIP-1-alpha release from isolated mononuclear cells of patients with severe sepsis. Inflamm Res 2004; 53: 528-533.
• 12 Brueckmann M, Horn S, Lang S. et al. Recombinant human activated protein C upregulates cyclooxygenase- 2 expression in endothelial cells via binding to endothelial cell protein C receptor and activation of PAR-1. Thromb Haemost 2005; 93: 743-750.
• 13 Yuksel M, Okajima K, Uchiba M. et al. Activated protein C inhibits lipopolysaccharide-induced tumor necrosis factor-alpha production by inhibiting activation of both nuclear factor-kappa B and activator protein- 1 in human monocytes. Thromb Haemost 2002; 88: 267-273.
• 14 Kaminska B. MAPK signalling pathways as molecular targets for anti-inflammatory therapy – from molecular mechanisms to therapeutic benefits. Biochim Biophys Acta 2005; 1754: 253-262.
• 15 Schieven GL. The biology of p38 kinase: a central role in inflammation. Curr Top Med Chem 2005; 5: 921-928.
• 16 Cain BS, Meldrum DR, Meng X. et al. p38 MAPK inhibition decreases TNF-alpha production and enhances postischemic human myocardial function. J Surg Res 1999; 83: 7-12.
• 17 Westra J, Limburg PC, de Boer P. et al. Effects of RWJ 67657, a p38 mitogen activated protein kinase (MAPK) inhibitor, on the production of inflammatory mediators by rheumatoid synovial fibroblasts. Ann Rheum Dis 2004; 63: 1453-1459.
• 18 Badger AM, Bradbeer JN, Votta B. et al. Pharmacological profile of SB 203580, a selective inhibitor of cytokine suppressive binding protein/p38 kinase, in animal models of arthritis, bone resorption, endotoxin shock and immune function. J Pharmacol Exp Ther 1996; 279: 1453-1461.
• 19 Song GY, Chung CS, Chaudry IH. et al. MAPK p38 antagonism as a novel method of inhibiting lymphoid immune suppression in polymicrobial sepsis. Am J Physiol Cell Physiol 2001; 281: C662-669.
• 20 Hommes D, van den Blink B, Plasse T. et al. Inhibition of stress-activated MAP kinases induces clinical improvement in moderate to severe Crohn’s disease. Gastroenterology 2002; 122: 7-14.
• 21 Branger J, van den Blink B, Weijer S. et al. Anti-inflammatory effects of a p38 mitogen-activated protein kinase inhibitor during human endotoxemia. J Immunol 2002; 168: 4070-4077.
• 22 Esmon CT. Role of coagulation inhibitors in inflammation. Thromb Haemost 2001; 86: 51-56.
• 23 Veldman A, Fischer D. The search for a unified theory of coagulation and inflammation. Cell Mol Life Sci 2004; 61: 2744-2749.
• 24 Leist M, Gantner F, Bohlinger I. et al. Murine hepatocyte apoptosis induced in vitro and in vivo by TNFalpha requires transcriptional arrest. J Immunol 1994; 153: 1778-1788.
• 25 Laszik Z, Mitro A, Taylor Jr. FB. et al. Human protein C receptor is present primarily on endothelium of large blood vessels: implications for the control of the protein C pathway. Circulation 1997; 96: 3633-3640.
• 26 Macias WL, Yan SB, Williams MD. et al. New insights into the protein C pathway: potential implications for the biological activities of drotrecogin alfa (activated). Crit Care 2005; 9: S38-45.
• 27 Feistritzer C, Mosheimer BA, Sturn DH. et al. Endothelial protein C receptor-dependent inhibition of migration of human lymphocytes by protein C involves epidermal growth factor receptor. J Immunol 2006; 176: 1019-1025.
• 28 Esmon CT. Thrombomodulin as a model of molecular mechanisms that modulate protease specificity and function at the vessel surface. Faseb J 1995; 9: 946-955.
• 29 Gomez-Lazaro M, Fernandez-Gomez FJ, Jordan J. p53: twenty five years understanding the mechanism of genome protection. J Physiol Biochem 2004; 60: 287-307.
• 30 Feistritzer C, Riewald M. Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine1-phosphate receptor-1 crossactivation. Blood 2005; 105: 3178-3184.
• 31 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.
• 32 Garcia JG, Patterson C, Bahler C. et al. Thrombin receptor activating peptides induce Ca2+ mobilization, barrier dysfunction, prostaglandin synthesis, and platelet- derived growth factor mRNA expression in cultured endothelium. J Cell Physiol 1993; 156: 541-549.
• 33 Garcia JG, Liu F, Verin AD. et al. Sphingosine 1-phosphate promotes endothelial cell barrier integrity by Edg-dependent cytoskeletal rearrangement. J Clin Invest 2001; 108: 689-701.
• 34 Annane D, Sebille V, Bellissant E. et al. Effect of low doses of corticosteroids in septic shock patients with or without early acute respiratory distress syndrome. Crit Care Med 2006; 34: 22-30.
• 35 Muhl H, Pfeilschifter J. Controlling the cytokine storm by insulin: glycogen synthase kinase-3 as a target in systemic inflammation. Crit Care Med 2006; 34: 1567-1569.
• 36 Docke WD, Randow F, Syrbe U. et al. Monocyte deactivation in septic patients: restoration by IFNgamma treatment. Nat Med 1997; 3: 678-681.
• 37 van den Berghe G, Wouters P, Weekers F. et al. Intensive insulin therapy in the critically ill patients. N Engl J Med 2001; 345: 1359-1367.
• 38 Kopp CW, Grey ST, Siegel JB. et al. Expression of human thrombomodulin cofactor activity in porcine endothelial cells. Transplantation 1998; 66: 244-251.
• 39 Faust SN, Levin M, Harrison OB. et al. Dysfunction of endothelial protein C activation in severe meningococcal sepsis. N Engl J Med 2001; 345: 408-416.
• 40 de Kleijn ED, de Groot R, Hack CE. et al. Activation of protein C following infusion of protein C concentrate in children with severe meningococcal sepsis and purpura fulminans: a randomized, double-blinded, placebo-controlled, dose-finding study. Crit Care Med 2003; 31: 1839-1847.
• 41 Spiel AO, Firbas C, Mayr FB. et al. The effects of supra-normal protein C levels on markers of coagulation, fibrinolysis and inflammation in a human model of endotoxemia. Thromb Haemost 2005; 94: 1148-1155.
• 42 Delvos U, Meusel P, Preissner KT. et al. Formation of activated protein C and inactivation of cell-bound thrombin by antithrombin III at the surface of cultured vascular endothelial cells--a comparative study of two anticoagulant mechanisms. Thromb Haemost 1987; 57: 87-91.
• 43 Taylor Jr., FB, Chang A, Esmon CT. et al. Protein C prevents the coagulopathic and lethal effects of Escherichia coli infusion in the baboon. J Clin Invest 1987; 79: 918-925.
• 44 Fourrier F, Jourdain M, Tournoys A. et al. Effects of a combined antithrombin III and protein C supplementation in porcine acute endotoxic shock. Shock 1998; 10: 364-370.
• 45 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.
• 46 Levi M, Dorffler-Melly J, Reitsma P. et al. Aggravation of endotoxin-induced disseminated intravascular coagulation and cytokine activation in heterozygous protein-C-deficient mice. Blood 2003; 101: 4823-4827.
• 47 Machino T, Hashimoto S, Maruoka S. et al. Apoptosis signal-regulating kinase 1-mediated signaling pathway regulates hydrogen peroxide-induced apoptosis in human pulmonary vascular endothelial cells. Crit Care Med 2003; 31: 2776-2781.
• 48 Lee JC, Laydon JT, McDonnell PC. et al. A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature 1994; 372: 739-746.
• 49 Song GY, Chung CS, Jarrar D. et al. Evolution of an immune suppressive macrophage phenotype as a product of P38 MAPK activation in polymicrobial sepsis. Shock 2001; 15: 42-48.
• 50 Hirose K, Okajima K, Taoka Y. et al. Activated protein C reduces the ischemia/reperfusion-induced spinal cord injury in rats by inhibiting neutrophil activation. Ann Surg 2000; 232: 272-280.
• 51 Nick JA, Coldren CD, Geraci MW. et al. Recombinant human activated protein C reduces human endotoxin- induced pulmonary inflammation via inhibition of neutrophil chemotaxis. Blood 2004; 104: 3878-3885.
• 52 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.
• 53 Wang M, Sankula R, Tsai BM. et al. P38 MAPK mediates myocardial proinflammatory cytokine production and endotoxin-induced contractile suppression. Shock 2004; 21: 170-174.
• 54 Xue M, Campbell D, Sambrook PN. et al. Endothelial protein C receptor and protease-activated receptor- 1 mediate induction of a wound-healing phenotype in human keratinocytes by activated protein C. J Invest Dermatol 2005; 125: 1279-1285.
• 55 Munshi N, Fernandis AZ, Cherla RP. et al. LPS-induced apoptosis of endothelial cells and its inhibition by vascular endothelial growth factor. J Immunol 2002; 168: 5860-5866.
• 56 Murphy FJ, Hayes I, Cotter TG. Targeting inflammatory diseases via apoptotic mechanisms. Curr Opin Pharmacol 2003; 3: 412-419.
• 57 Bannerman DD, Sathyamoorthy M, Goldblum SE. Bacterial lipopolysaccharide disrupts endothelial monolayer integrity and survival signaling events through caspase cleavage of adherens junction proteins. J Biol Chem 1998; 273: 35371-35380.
• 58 Hotchkiss RS, Swanson PE, Freeman BD. et al. Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction. Crit Care Med 1999; 27: 1230-1251.
• 59 Mayr M, Hu Y, Hainaut H. et al. Mechanical stressinduced DNA damage and rac-p38MAPK signal pathways
• 60 Mannick JA. Trauma, sepsis and immune defects. In: Faist E, Meakins J. L., Schildberg FW. ed. Host Defense Dysfunction in Trauma, Shock and Sepsis. Berlin:: Springer-Verlag; 1993: 15-21.