, Volume 14, Issue 4, pp 503–513

Chronic hypoxia impairs extracellular nucleotide metabolism and barrier function in pulmonary artery vasa vasorum endothelial cells

  • Gennady G. Yegutkin
  • Mikko Helenius
  • Elzbieta Kaczmarek
  • Nana Burns
  • Sirpa Jalkanen
  • Kurt Stenmark
  • Evgenia V. Gerasimovskaya
Original Paper


Vascular remodeling plays a pivotal role in a variety of pathophysiological conditions where hypoxia and inflammation are prominent features. Intravascular ATP, ADP and adenosine are known as important regulators of vascular tone, permeability and homeostasis, however contribution of purinergic signalling to endothelial cell growth and angiogenesis remains poorly understood. By using vasa vasorum endothelial cells (VVEC) isolated from pulmonary artery adventitia of control and chronically hypoxic neonatal calves, these studies were aimed to evaluate the effect of hypoxia on biochemical and functional properties of microvascular endothelial network at the sites of angiogenesis. In comparison with normoxic controls, VVEC from hypoxic animals are characterized by (1) drastically impaired nucleoside triphosphate diphosphohydrolase-1 (NTPDase-1/CD39) and ecto-5′-nucleotidase/CD73 activities with respective increases in basal extracellular ATP and ADP levels (2) higher proliferative responses to low micromolar concentrations of ATP and ADP; and (3) enhanced permeability and disordered adenosinergic control of vascular barrier function (measured as a paracellular flux of 70 kDa fluorescein isothiocyanate-dextran). Together, these results suggest that unique pattern of purine-mediated angiogenic activation and enhanced leakiness of VVEC from chronically hypoxic vessels may be defined by disordered endothelial nucleotide homeostasis at sites of active neovascularization.


Purinergic signaling Endothelial cells Vasa vasorum Hypoxia NTPDase Ecto-5′-nucleotidase 


  1. 1.
    Robson SC, Wu Y, Sun X, Knosalla C, Dwyer K, Enjyoji K (2005) Ectonucleotidases of CD39 family modulate vascular inflammation and thrombosis in transplantation. Semin Thromb Hemost 31:217–233PubMedCrossRefGoogle Scholar
  2. 2.
    Stenmark KR, Fagan KA, Frid MG (2006) Hypoxia-induced pulmonary vascular remodeling: cellular and molecular mechanisms. Circ Res 99:675–691PubMedCrossRefGoogle Scholar
  3. 3.
    Morrell NW, Adnot S, Archer SL, Dupuis J, Jones PL, MacLean MR, McMurtry IF, Stenmark KR, Thistlethwaite PA, Weissmann N, Yuan JX, Weir EK (2009) Cellular and molecular basis of pulmonary arterial hypertension. J Am Coll Cardiol 54:S20–S31PubMedCrossRefGoogle Scholar
  4. 4.
    Bours MJL, Swennen ELR, Di Virgilio F, Cronstein BN, Dagnelie PC (2006) Adenosine 5′-triphosphate and adenosine as endogenous signaling molecules in immunity and inflammation. Pharmacol Ther 112:358–404PubMedCrossRefGoogle Scholar
  5. 5.
    Erlinge D, Burnstock G (2008) P2 receptors in cardiovascular regulation and disease. Purinergic Signal 4:1–20PubMedCrossRefGoogle Scholar
  6. 6.
    Kaczmarek E, Erb L, Koziak K, Jarzyna R, Wink MR, Guckelberger O, Blusztajn JK, Trinkaus-Randall V, Weisman GA, Robson SC (2005) Modulation of endothelial cell migration by extracellular nucleotides: involvement of focal adhesion kinase and phosphatidylinositol 3-kinase-mediated pathways. Thromb Haemost 93:735–742PubMedGoogle Scholar
  7. 7.
    Gerasimovskaya EV, Woodward HN, Tucker DA, Stenmark KR (2008) Extracellular ATP is a pro-angiogenic factor for pulmonary artery vasa vasorum endothelial cells. Angiogenesis 11:169–182PubMedCrossRefGoogle Scholar
  8. 8.
    Rumjahn SM, Yokdang N, Baldwin KA, Thai J, Buxton IL (2009) Purinergic regulation of vascular endothelial growth factor signaling in angiogenesis. Br J Cancer 100:1465–1470PubMedCrossRefGoogle Scholar
  9. 9.
    Ralevic V, Burnstock G (1998) Receptors for purines and pyrimidines. Pharmacol Rev 50:413–492PubMedGoogle Scholar
  10. 10.
    Yegutkin GG (2008) Nucleotide- and nucleoside-converting ectoenzymes: important modulators of purinergic signalling cascade. Biochim Biophys Acta 1783:673–694PubMedCrossRefGoogle Scholar
  11. 11.
    Kaczmarek E, Koziak K, Sevigny J, Siegel JB, Anrather J, Beaudoin AR, Bach FH, Robson SC (1996) Identification and characterization of CD39/vascular ATP diphosphohydrolase. J Biol Chem 271:33116–33122PubMedCrossRefGoogle Scholar
  12. 12.
    Robson SC, Sevigny J, Zimmermann H (2006) The E-NTPDase family of ectonucleotidases: structure function relationship and pathophysiological significance. Purinergic Signal 2:409–430PubMedCrossRefGoogle Scholar
  13. 13.
    Goepfert C, Sundberg C, Sevigny J, Enjyoji K, Hoshi T, Csizmadia E, Robson S (2001) Disordered cellular migration and angiogenesis in cd39-null mice. Circulation 104:3109–3115PubMedCrossRefGoogle Scholar
  14. 14.
    Jackson SW, Hoshi T, Wu Y, Sun X, Enjyoji K, Cszimadia E, Sundberg C, Robson SC (2007) Disordered purinergic signaling inhibits pathological angiogenesis in cd39/Entpd1-null mice. Am J Pathol 171:1395–1404PubMedCrossRefGoogle Scholar
  15. 15.
    Zernecke A, Bidzhekov K, Ozuyaman B, Fraemohs L, Liehn EA, Luscher-Firzlaff JM, Luscher B, Schrader J, Weber C (2006) CD73/ecto-5′-nucleotidase protects against vascular inflammation and neointima formation. Circulation 113:2120–2127PubMedCrossRefGoogle Scholar
  16. 16.
    Kiss J, Yegutkin GG, Koskinen K, Savunen T, Jalkanen S, Salmi M (2007) IFN-beta protects from vascular leakage via up-regulation of CD73. Eur J Immunol 37:3334–3338PubMedCrossRefGoogle Scholar
  17. 17.
    Jacobson KA, Gao ZG (2006) Adenosine receptors as therapeutic targets. Nat Rev Drug Discov 5:247–264PubMedCrossRefGoogle Scholar
  18. 18.
    Woodward HN, Anwar A, Riddle S, Taraseviciene-Stewart L, Fragoso M, Stenmark KR, Gerasimovskaya EV (2009) PI3 K, Rho, and ROCK play a key role in hypoxia-induced ATP release and ATP-stimulated angiogenic responses in pulmonary artery vasa vasorum endothelial cells. Nat Rev Drug Discov 297:L954–L964Google Scholar
  19. 19.
    Lyubchenko T, Woodward H, Veo KD, Burns N, Nijmeh H, Liubchenko GA, Stenmark KR, Gerasimovskaya EV (2011) P2Y1 and P2Y13 purinergic receptors mediate Ca2 + signaling and proliferative responses in pulmonary artery vasa vasorum endothelial cells. Am J Physiol Cell Physiol 300:C266–C275PubMedCrossRefGoogle Scholar
  20. 20.
    Yegutkin GG, Henttinen T, Jalkanen S (2001) Extracellular ATP formation on vascular endothelial cells is mediated by ecto-nucleotide kinase activities via phosphotransfer reactions. FASEB J 15:251–260PubMedCrossRefGoogle Scholar
  21. 21.
    Yegutkin GG, Samburski SS, Jalkanen S (2003) Soluble purine-converting enzymes circulate in human blood and regulate extracellular ATP level via counteracting pyrophosphatase and phosphotransfer reactions. Faseb J 17:1328–1330PubMedGoogle Scholar
  22. 22.
    Muller CE, Iqbal J, Baqi Y, Zimmermann H, Rollich A, Stephan H (2006) Polyoxometalates—a new class of potent ecto-nucleoside triphosphate diphosphohydrolase (NTPDase) inhibitors. Bioorg Med Chem Lett 16:5943–5947PubMedCrossRefGoogle Scholar
  23. 23.
    Henttinen T, Jalkanen S, Yegutkin GG (2003) Adherent leukocytes prevent adenosine formation and impair endothelial barrier function by Ecto-5′-nucleotidase/CD73-dependent mechanism. J Biol Chem 278:24888–24895PubMedCrossRefGoogle Scholar
  24. 24.
    Colgan SP, Eltzschig HK, Eckle T, Thompson LF (2006) Physiological roles for ecto-5′-nucleotidase (CD73). Purinergic Signal 2:351–360PubMedCrossRefGoogle Scholar
  25. 25.
    Carpenter TC, Stenmark KR (2001) Hypoxia decreases lung neprilysin expression and increases pulmonary vascular leak. Am J Physiol Lung Cell Mol Physiol 281:L941–L948PubMedGoogle Scholar
  26. 26.
    Thompson LF, Eltzschig HK, Ibla JC, Van De Wiele CJ, Resta R, Morote-Garcia JC, Colgan SP (2004) Crucial role for ecto-5’-nucleotidase (CD73) in vascular leakage during hypoxia. J Exp Med 200:1395–1405PubMedCrossRefGoogle Scholar
  27. 27.
    Sayner SL (2011) Emerging themes of cAMP regulation of the pulmonary endothelial barrier. Am J Physiol Lung Cell Mol Physiol 300:L667–L678PubMedCrossRefGoogle Scholar
  28. 28.
    Lazarowski ER, Tarran R, Grubb BR, van Heusden CA, Okada S, Boucher RC (2004) Nucleotide release provides a mechanism for airway surface liquid homeostasis. J Biol Chem 279:36855–36864PubMedCrossRefGoogle Scholar
  29. 29.
    Gerasimovskaya EV, Stenmark KR, Yegutkin GG (2010) Role of purine-converting ecto-enzymes in angiogenic phenotype of pulmonary artery adventitial vasa vasorum endothelial cells of chronically hypoxic calves. In: Gerasimovskaya EV, Kaczmarek E (eds) Extracellular ATP and adenosine as regulators of endothelial cell function. Springer, Dordrecht, Netherlands, pp 73–93CrossRefGoogle Scholar
  30. 30.
    Robson SC, Kaczmarek E, Siegel JB, Candinas D, Koziak K, Millan M, Hancock WW, Bach FH (1997) Loss of ATP diphosphohydrolase activity with endothelial cell activation. J Exp Med 185:153–163PubMedCrossRefGoogle Scholar
  31. 31.
    Gerasimovskaya EV, Ahmad S, White CW, Jones PL, Carpenter TC, Stenmark KR (2002) Extracellular ATP is an autocrine/paracrine regulator of hypoxia-induced adventitial fibroblast growth. Signaling through extracellular signal-regulated kinase-1/2 and the Egr-1 transcription factor. J Biol Chem 277:44638–44650PubMedCrossRefGoogle Scholar
  32. 32.
    Eltzschig HK, Ibla JC, Furuta GT, Leonard MO, Jacobson KA, Enjyoji K, Robson SC, Colgan SP (2003) Coordinated adenine nucleotide phosphohydrolysis and nucleoside signaling in posthypoxic endothelium: role of ectonucleotidases and adenosine A2B receptors. J Exp Med 198:783–796PubMedCrossRefGoogle Scholar
  33. 33.
    Kobayashi S, Zimmermann H, Millhorn DE (2000) Chronic hypoxia enhances adenosine release in rat PC12 cells by altering adenosine metabolism and membrane transport. J Neurochem 74:621–632PubMedCrossRefGoogle Scholar
  34. 34.
    Eltzschig HK, Faigle M, Knapp S, Karhausen J, Ibla J, Rosenberger P, Odegard KC, Laussen PC, Thompson LF, Colgan SP (2006) Endothelial catabolism of extracellular adenosine during hypoxia: the role of surface adenosine deaminase and CD26. Blood 108:1602–1610PubMedCrossRefGoogle Scholar
  35. 35.
    Hunsucker SA, Mitchell BS, Spychala J (2005) The 5′-nucleotidases as regulators of nucleotide and drug metabolism. Pharmacol Ther 107:1–30PubMedCrossRefGoogle Scholar
  36. 36.
    Narravula S, Lennon PF, Mueller BU, Colgan SP (2000) Regulation of endothelial CD73 by adenosine: paracrine pathway for enhanced endothelial barrier function. J Immunol 165:5262–5268PubMedGoogle Scholar
  37. 37.
    Stout JG, Kirley TL (1996) Control of cell membrane ecto-ATPase by oligomerization state: intermolecular cross-linking modulates ATPase activity. Biochemistry 35:8289–8298PubMedCrossRefGoogle Scholar
  38. 38.
    Knowles AF (2011) The GDA1_CD39 superfamily: NTPDases with diverse functions. Purinergic Signal 7:21–45PubMedCrossRefGoogle Scholar
  39. 39.
    Yegutkin GG, Mikhailov A, Samburski SS, Jalkanen S (2006) The detection of micromolar pericellular ATP pool on lymphocyte surface by using lymphoid ecto-adenylate kinase as intrinsic ATP sensor. Mol Biol Cell 17:3378–3385PubMedCrossRefGoogle Scholar
  40. 40.
    Kolosova IA, Mirzapoiazova T, Adyshev D, Usatyuk P, Romer LH, Jacobson JR, Natarajan V, Pearse DB, Garcia JG, Verin AD (2005) Signaling pathways involved in adenosine triphosphate-induced endothelial cell barrier enhancement. Circ Res 97:115–124PubMedCrossRefGoogle Scholar
  41. 41.
    Hasegawa T, Bouis D, Liao H, Visovatti SH, Pinsky DJ (2008) Ecto-5′ nucleotidase (CD73)-mediated adenosine generation and signaling in murine cardiac allograft vasculopathy. Circ Res 103:1410–1421PubMedCrossRefGoogle Scholar
  42. 42.
    Eckle T, Faigle M, Grenz A, Laucher S, Thompson LF, Eltzschig HK (2008) A2B adenosine receptor dampens hypoxia-induced vascular leak. Blood 111:2024–2035PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2011

Authors and Affiliations

  • Gennady G. Yegutkin
    • 1
  • Mikko Helenius
    • 1
  • Elzbieta Kaczmarek
    • 2
  • Nana Burns
    • 3
  • Sirpa Jalkanen
    • 1
  • Kurt Stenmark
    • 3
  • Evgenia V. Gerasimovskaya
    • 3
  1. 1.MediCity Research LaboratoryUniversity of Turku and National Institute of Health and WelfareTurkuFinland
  2. 2.Beth Israel Deaconess Medical CenterHarvard Medical School and Center for Vascular Biology ResearchBostonUSA
  3. 3.Department of PediatricsUniversity of Colorado DenverAuroraUSA

Personalised recommendations