Cellular and Molecular Life Sciences

, Volume 67, Issue 9, pp 1537–1546

Endogenous protein C is essential for the functional integrity of human endothelial cells

  • Meilang Xue
  • Nikita Minhas
  • Shu-Oi Chow
  • Suat Dervish
  • Philip N. Sambrook
  • Lyn March
  • Christopher J. Jackson
Research Article

Abstract

Circulating protein C (PC) plays a vital role as an anti-coagulant and anti-inflammatory mediator. We show here that human endothelial cells produce PC that acts through novel mediators to enhance their own functional integrity. When endogenous PC or its receptor, endothelial protein C receptor (EPCR), was suppressed by small interfering (si) RNA, human umbilical cord endothelial cell (HUVEC) proliferation was decreased and apoptosis elevated. Interestingly, PC or EPCR siRNA significantly increased HUVEC permeability, which is likely via reduction of the angiopoietin (Ang)1/Ang2 ratio and inhibition of the peripheral localization of the tight junction protein, zona occludins-1. In addition, PC or EPCR siRNA inhibited type IV collagen and matrix metalloproteinase-2, providing the first evidence that PC contributes to vascular basement membrane formation. These newly described actions of endogenous PC act to stabilize endothelial cells and enhance barrier function, to potentially promote the functional integrity of blood vessels.

Keywords

Activated protein C Endothelial protein C receptor Permeability Angiopoietin 

Abbreviations

APC

Activated protein C

EPCR

Endothelial protein C receptor

HUVEC

Human umbilical cord endothelial cells

MMP

Matrix metalloproteinase

siRNA

Small interfering RNA

ZO-1

Zonula occludens-1

References

  1. 1.
    Fukudome K, Ye XF, Tsuneyoshi N, Tokunaga O, Sugawara K, Mizokami H, Kimoto M (1998) Activation mechanism of anticoagulant protein C in large blood vessels involving the endothelial cell protein c receptor. J Exp Med 187:1029–1035CrossRefPubMedGoogle Scholar
  2. 2.
    Stearns-Kurosawa DJ, Kurosawa S, Mollica JS, Ferrell GL, Esmon CT (1996) The endothelial cell protein C receptor augments protein C activation by the thrombin–thrombomoduliná complex. Proc Natl Acad Sci USA 93:10212–10216CrossRefPubMedGoogle Scholar
  3. 3.
    Fukudome K, Esmon CT (1994) Identification, cloning, and regulation of a novel endothelial cell protein c activated protein c receptor. J Biol Chem 269:26486–26491PubMedGoogle Scholar
  4. 4.
    Villoutreix BO, Blom AM, Dahlback B (1999) Structural prediction and analysis of endothelial cell protein C/activated protein C receptor. Protein Eng 12:833–840CrossRefPubMedGoogle Scholar
  5. 5.
    Xu J, Esmon NL, Esmon CT (1999) Reconstitution of the human endothelial cell protein C receptor with thrombomodulin in phosphatidylcholine vesicles enhances protein C activation. J Biol Chem 274:6704–6710CrossRefPubMedGoogle Scholar
  6. 6.
    Baker WF Jr, Bick RL (1999) Treatment of hereditary and acquired thrombophilic disorders. Semin Thromb Hemost 25:387–405CrossRefPubMedGoogle Scholar
  7. 7.
    Griffin JH, Fernandez JA, Mosnier LO, Liu D, Cheng T, Guo H, Zlokovic BV (2006) The promise of protein C. Blood Cells Mol Dis 36:211–216CrossRefPubMedGoogle Scholar
  8. 8.
    Jackson CJ, Xue M (2008) Activated protein C-an anticoagulant that does more than stop clots. Int J Biochem Cell Biol 40:2692–2697CrossRefPubMedGoogle Scholar
  9. 9.
    Feistritzer C, Riewald M (2005) Endothelial barrier protection by activated protein C through PAR1-dependent sphingosine 1-phosphate receptor-1 crossactivation. Blood 105:3178–3184CrossRefPubMedGoogle Scholar
  10. 10.
    Finigan JH, Dudek SM, Singleton PA, Chiang ET, Jacobson JR, Camp SM, Ye SQ, Garcia JG (2005) Activated protein C mediates novel lung endothelial barrier enhancement: role of sphingosine 1-phosphate receptor transactivation. J Biol Chem 280:17286–17293CrossRefPubMedGoogle Scholar
  11. 11.
    Feistritzer C, Schuepbach RA, Mosnier LO, Bush LA, Di CE, Griffin JH, Riewald M (2006) Protective signaling by activated protein C is mechanistically linked to protein C activation on endothelial cells. J Biol Chem 281:20077–20084CrossRefPubMedGoogle Scholar
  12. 12.
    Nguyen M, Arkell J, Jackson CJ (2000) Activated protein C directly activates human endothelial gelatinase A. J Biol Chem 275:9095–9098CrossRefPubMedGoogle Scholar
  13. 13.
    Herron GS, Banda MJ, Clark EJ, Gavrilovic J, Werb Z (1986) Secretion of metalloproteinases by stimulated capillary endothelial cells II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors. J Biol Chem 261:2814–2818PubMedGoogle Scholar
  14. 14.
    Patterson CE, Rhoades RA, Garcia JG (1992) Evans blue dye as a marker of albumin clearance in cultured endothelial monolayer and isolated lung. J Appl Physiol 72:865–873CrossRefPubMedGoogle Scholar
  15. 15.
    Uchiba M, Okajima K, Oike Y, Ito Y, Fukudome K, Isobe H, Suda T (2004) Activated protein C induces endothelial cell proliferation by mitogen-activated protein kinase activation in vitro and angiogenesis in vivo. Circ Res 95:34–41CrossRefPubMedGoogle Scholar
  16. 16.
    Fukuhara S, Sako K, Minami T, Noda K, Kim HZ, Kodama T, Shibuya M, Takakura N, Koh GY, Mochizuki N (2008) Differential function of Tie2 at cell-cell contacts and cell-substratum contacts regulated by angiopoietin-1. Nat Cell Biol 10:513–526CrossRefPubMedGoogle Scholar
  17. 17.
    Saharinen P, Eklund L, Miettinen J, Wirkkala R, Anisimov A, Winderlich M, Nottebaum A, Vestweber D, Deutsch U, Koh GY, Olsen BR, Alitalo K (2008) Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell–cell and cell–matrix contacts. Nat Cell Biol 10:527–537CrossRefPubMedGoogle Scholar
  18. 18.
    Makinde T, Agrawal DK (2008) Intra and extravascular transmembrane signalling of angiopoietin-1-Tie2 receptor in health and disease. J Cell Mol Med 12:810–828CrossRefPubMedGoogle Scholar
  19. 19.
    Salmon AHJ, Neal CR, Sage LM, Glass CA, Harper SJ, Bates DO (2009) Angiopoietin-1 alters microvascular permeability coefficients in vivo via modification of endothelial glycocalyx. Cardiovasc Res 83:24–33CrossRefPubMedGoogle Scholar
  20. 20.
    Wong AL (1997) Tie2 expression and phosphorylation in angiogenic and quiescent adult tissues. Circ Res 81:567–574PubMedGoogle Scholar
  21. 21.
    Timpl R (1996) Macromolecular organization of basement membranes. Curr Opin Cell Biol 8:618–624CrossRefPubMedGoogle Scholar
  22. 22.
    Page-McCaw A, Ewald AJ, Werb Z (2007) Matrix metalloproteinases and the regulation of tissue remodelling. Nat Rev Mol Cell Biol 8:221–233CrossRefPubMedGoogle Scholar
  23. 23.
    Xue M, Le NT, Jackson CJ (2006) Targeting matrix metalloproteases to improve cutaneous wound healing. Expert Opin Ther Targets 10:143–155CrossRefPubMedGoogle Scholar
  24. 24.
    Xue M, Thompson P, Sambrook PN, March L, Jackson CJ (2006) Activated protein C stimulates expression of angiogenic factors in human skin cells, angiogenesis in the chick embryo and cutaneous wound healing in rodents. Clin Hemorheol Microcirc 34:153–161PubMedGoogle Scholar
  25. 25.
    Tanabe S, Sugo T, Matsuda M (1991) Synthesis of protein C in human umbilical vein endothelial cells. J Biochem 109:924–928PubMedGoogle Scholar
  26. 26.
    Laszik Z, Mitro A, Taylor FB, Ferrell G, Esmon CT (1997) Human protein c receptor is present primarily on endothelium of large blood vessels: implications for the control of the protein c pathway. Circulation 96:3633–3640PubMedGoogle Scholar
  27. 27.
    Xue M, Campbell D, Jackson CJ (2007) Protein C is an autocrine growth factor for human skin keratinocytes. J Biol Chem 282:13610–13616CrossRefPubMedGoogle Scholar
  28. 28.
    Liu X, Zou H, Slaughter C, Wang X (1997) DFF, a heterodimeric protein that functions downstream of caspase-3 to trigger DNA fragmentation during apoptosis. Cell 89:175–184CrossRefPubMedGoogle Scholar
  29. 29.
    Sakahira H, Enari M, Nagata S (1998) Cleavage of CAD inhibitor in CAD activation and DNA degradation during apoptosis. Nature 391:96–99CrossRefPubMedGoogle Scholar
  30. 30.
    Chandler JM, Cohen GM, MacFarlane M (1998) Different subcellular distribution of caspase-3 and caspase-7 following Fas-induced apoptosis in mouse liver. J Biol Chem 273:10815–10818CrossRefPubMedGoogle Scholar
  31. 31.
    Fanning AS, Jameson BJ, Jesaitis LA, Anderson JM (1998) The tight junction protein ZO-1 establishes a link between the transmembrane protein occludin and the actin cytoskeleton. J Biol Chem 273:29745–29753CrossRefPubMedGoogle Scholar
  32. 32.
    Musch MW, Walsh-Reitz MM, Chang EB (2006) Roles of ZO-1, occludin, and actin in oxidant-induced barrier disruption. Am J Physiol Gastrointest Liver Physiol 290:G222–G231CrossRefPubMedGoogle Scholar
  33. 33.
    Anderson JM, Van Itallie CM, Peterson MD, Stevenson BR, Carew EA, Mooseker MS (1989) ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells. J Cell Biol 109:1047–1056CrossRefPubMedGoogle Scholar
  34. 34.
    Riewald M, Petrovan RJ, Donner A, Mueller BM, Ruf W (2002) Activation of endothelial cell protease activated receptor 1 by the protein C pathway. Science 296:1880–1882CrossRefPubMedGoogle Scholar
  35. 35.
    Bae JS, Yang L, Manithody C, Rezaie AR (2007) Engineering a disulfide bond to stabilize the calcium binding loop of activated protein C eliminates its anticoagulant but not protective signaling properties. J Biol Chem 282:9251–9259CrossRefPubMedGoogle Scholar
  36. 36.
    Bae JS, Yang L, Rezaie AR (2007) Receptors of the protein C activation and activated protein C signaling pathways are colocalized in lipid rafts of endothelial cells. Proc Natl Acad Sci USA 104(8):2867–2872CrossRefPubMedGoogle Scholar
  37. 37.
    Minhas N, Xue M, Fukudome K, Jackson CJ (2009) Activated protein C utilizes the angiopoietin/Tie2 axis to promote endothelial barrier function. FASEB J PMID: 19858095 (Epub ahead of print)Google Scholar
  38. 38.
    Gu JM, Crawley JTB, Ferrell G, Zhang F, Li W, Esmon NL, Esmon CT (2002) Disruption of the endothelial cell protein C receptor gene in mice causes placental thrombosis and early embryonic lethality. J Biol Chem 277:43335–43343CrossRefPubMedGoogle Scholar
  39. 39.
    Jalbert LR, Rosen ED, Moons L, Chan JC, Carmeliet P, Collen D, Castellino FJ (1998) Inactivation of the gene for anticoagulant protein C causes lethal perinatal consumptive coagulopathy in mice. J Clin Invest 102:1481–1488CrossRefPubMedGoogle Scholar
  40. 40.
    Castellino FJ, Liang Z, Volkir SP, Haalboom E, Martin JA, Sandoval-Cooper MJ, Rosen ED (2002) Mice with a severe deficiency of the endothelial protein C receptor gene develop, survive, and reproduce normally, and do not present with enhanced arterial thrombosis after challenge. Thromb Haemost 88:462–472PubMedGoogle Scholar
  41. 41.
    Lay AJ, Liang Z, Rosen ED, Castellino FJ (2005) Mice with a severe deficiency in protein C display prothrombotic and proinflammatory phenotypes and compromised maternal reproductive capabilities. J Clin Invest 115:1552–1561CrossRefPubMedGoogle Scholar
  42. 42.
    Wang YL, Hui YN, Guo B, Ma JX (2007) Strengthening tight junctions of retinal microvascular endothelial cells by pericytes under normoxia and hypoxia involving angiopoietin-1 signal way. Eye 21:1501–1510CrossRefPubMedGoogle Scholar
  43. 43.
    Peters KG (2004) Functional significance of Tie2 signaling in the adult vasculature. Recent Prog Horm Res 59:51–71CrossRefPubMedGoogle Scholar
  44. 44.
    LeBleu VS, Macdonald B, Kalluri R (2007) Structure and function of basement membranes. Exp Biol Med (Maywood) 232:1121–1129CrossRefGoogle Scholar
  45. 45.
    Sage H (1982) Collagens of basement membranes. J Invest Dermatol 79:51s–59sCrossRefPubMedGoogle Scholar
  46. 46.
    McQuibban GA, Gong JH, Wong JP, Wallace JL, Clark-Lewis I, Overall CM (2002) Matrix metalloproteinase processing of monocyte chemoattractant proteins generates CC chemokine receptor antagonists with anti-inflammatory properties in vivo. Blood 100:1160–1167PubMedGoogle Scholar
  47. 47.
    McQuibban GA, Gong JH, Tam EM, McCulloch CA, Clark-Lewis I, Overall CM (2000) Inflammation dampened by gelatinase A cleavage of monocyte chemoattractant protein-3. Science 289:1202–1206CrossRefPubMedGoogle Scholar

Copyright information

© Springer Basel AG 2010

Authors and Affiliations

  • Meilang Xue
    • 1
  • Nikita Minhas
    • 1
  • Shu-Oi Chow
    • 1
  • Suat Dervish
    • 1
  • Philip N. Sambrook
    • 2
  • Lyn March
    • 2
  • Christopher J. Jackson
    • 1
  1. 1.Sutton Research Laboratories, Institute of Bone and Joint Research, Kolling Institute of Medical ResearchThe University of Sydney at Royal North Shore HospitalSt LeonardsAustralia
  2. 2.Department of Rheumatology, Institute of Bone and Joint Research, Kolling Institute of Medical ResearchThe University of Sydney at Royal North Shore HospitalSt LeonardsAustralia

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