Purinergic P2Y2 receptors modulate endothelial sprouting

  • Severin Mühleder
  • Christiane Fuchs
  • José Basílio
  • Dorota Szwarc
  • Karoline Pill
  • Krystyna Labuda
  • Paul Slezak
  • Christian Siehs
  • Johannes Pröll
  • Eleni Priglinger
  • Carsten Hoffmann
  • Wolfgang G. Junger
  • Heinz Redl
  • Wolfgang HolnthonerEmail author
Original Article


Purinergic P2 receptors are critical regulators of several functions within the vascular system, including platelet aggregation, vascular inflammation, and vascular tone. However, a role for ATP release and P2Y receptor signalling in angiogenesis remains poorly defined. Here, we demonstrate that blood vessel growth is controlled by P2Y2 receptors. Endothelial sprouting and vascular tube formation were significantly dependent on P2Y2 expression and inhibition of P2Y2 using a selective antagonist blocked microvascular network generation. Mechanistically, overexpression of P2Y2 in endothelial cells induced the expression of the proangiogenic molecules CXCR4, CD34, and angiopoietin-2, while expression of VEGFR-2 was decreased. Interestingly, elevated P2Y2 expression caused constitutive phosphorylation of ERK1/2 and VEGFR-2. However, stimulation of cells with the P2Y2 agonist UTP did not influence sprouting unless P2Y2 was constitutively expressed. Finally, inhibition of VEGFR-2 impaired spontaneous vascular network formation induced by P2Y2 overexpression. Our data suggest that P2Y2 receptors have an essential function in angiogenesis, and that P2Y2 receptors present a therapeutic target to regulate blood vessel growth.


Endothelial Purinergic Angiogenesis P2Y2 Tip cell Sprouting 



The authors thank Johannes Zipperle for isolating human platelets and Regina Grillari for providing Phoenix Ampho cells. This work was funded in part by the European Union’s INTERREG V-A AT-CZ programme (ATCZ133), the City of Vienna Competence Team SignalTissue (#18-08) and by the Austrian Science Fund project SFB-F54. The funding sources have no influence on design and conduct of the study, collection, management, analysis and interpretation of the data, and preparation, review, or approval of the manuscript.

Author contributions

SM performed retroviral infections, spheroid and fibrin matrix assays, flow cytometry, RT-PCR, and immunoprecipitations. SM and KL generated retroviral plasmids. CF and DS performed immunoblotting. SM and KP performed proliferation assays and analyzed iPSC-ECFC in flow cytometry. JP generated the gene array data. CS and JB analyzed gene array data. EP and CH supported the study by providing material. PS, WJ, and HR co-advised the project. SM and WH designed the figures and wrote the manuscript. WH was the lead advisor of this work. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no competing interests.

Supplementary material

18_2019_3213_MOESM1_ESM.docx (1 mb)
Supplementary material 1 (DOCX 1051 kb)
18_2019_3213_MOESM2_ESM.avi (3.2 mb)
Supplementary material 2 (AVI 3292 kb) Supplementary movie 1: Z-scan image sequence of P2Y2OE-HUVEC embedded in a fibrin matrix assay in co-culture with MSC. The P2Y2-YFP fusion protein is localized in the cytoplasm, on cell–cell borders and on filopodia


  1. 1.
    Burnstock G, Ralevic V (2014) Purinergic signaling and blood vessels in health and disease. Pharmacol Rev 66:102–192. CrossRefPubMedGoogle Scholar
  2. 2.
    Eltzschig HK, Sitkovsky MV, Robson SC (2012) Purinergic signaling during inflammation. N Engl J Med 367:2322–2333. CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Wang L, Karlsson L, Moses S et al (2002) P2 receptor expression profiles in human vascular smooth muscle and endothelial cells. J Cardiovasc Pharmacol 40:841–853. CrossRefPubMedGoogle Scholar
  4. 4.
    Jin H, Seo J, Eun SY et al (2014) P2Y2 R activation by nucleotides promotes skin wound-healing process. Exp Dermatol 23:480–485. CrossRefPubMedGoogle Scholar
  5. 5.
    Gidlöf O, Sathanoori R, Magistri M et al (2015) Extracellular uridine triphosphate and adenosine triphosphate attenuate endothelial inflammation through miR-22-mediated ICAM-1 inhibition. J Vasc Res 52:71–80. CrossRefPubMedGoogle Scholar
  6. 6.
    Rumjahn SM, Yokdang N, Baldwin KA et al (2009) Purinergic regulation of vascular endothelial growth factor signaling in angiogenesis. Br J Cancer 100:1465–1470. CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Jacobson KA, Ivanov AA, de Castro S et al (2009) Development of selective agonists and antagonists of P2Y receptors. Purinergic Signal 5:75–89. CrossRefPubMedGoogle Scholar
  8. 8.
    Seye CI, Yu N, González FA et al (2004) The P2Y2 nucleotide receptor mediates vascular cell adhesion molecule-1 expression through interaction with VEGF receptor-2 (KDR/Flk-1). J Biol Chem 279:35679–35686. CrossRefPubMedGoogle Scholar
  9. 9.
    Liao Z, Cao C, Wang J et al (2014) The P2Y2 receptor interacts with VE-cadherin and VEGF receptor-2 to regulate Rac1 activity in endothelial cells. J Biomed Sci Eng 7:1105–1121. CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    McEnaney RM, Shukla A, Madigan MC et al (2016) P2Y2 nucleotide receptor mediates arteriogenesis in a murine model of hind limb ischemia. J Vasc Surg 63:216–225. CrossRefPubMedGoogle Scholar
  11. 11.
    Sivaraj KK, Li R, Albarran-Juarez J et al (2015) Endothelial Gαq/11 is required for VEGF-induced vascular permeability and angiogenesis. Cardiovasc Res 108:171–180. CrossRefPubMedGoogle Scholar
  12. 12.
    Liu J, Liao Z, Camden J et al (2004) Src homology 3 binding sites in the P2Y2 nucleotide receptor interact with Src and regulate activities of Src, proline-rich tyrosine kinase 2, and growth factor receptors. J Biol Chem 279:8212–8218. CrossRefPubMedGoogle Scholar
  13. 13.
    Erb L, Weisman GA (2015) Coupling of P2Y receptors to G proteins and other signaling pathways. Wiley Interdiscip Rev Membr Transp Signal 1:789–803. CrossRefGoogle Scholar
  14. 14.
    Wang S, Iring A, Strilic B et al (2015) P2Y2 and Gq/G11 control blood pressure by mediating endothelial mechanotransduction. J Clin Invest 125:3077–3086. CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Petzelbauer P, Bender JR, Wilson J, Pober JS (1993) Heterogeneity of dermal microvascular endothelial cell antigen expression and cytokine responsiveness in situ and in cell culture. J Immunol 151:5062–5072PubMedGoogle Scholar
  16. 16.
    Priglinger E, Maier J, Chaudary S et al (2018) Photobiomodulation of freshly isolated human adipose tissue-derived stromal vascular fraction cells by pulsed light-emitting diodes for direct clinical application. J Tissue Eng Regen Med 12:1352–1362. CrossRefPubMedGoogle Scholar
  17. 17.
    Sathanoori R, Bryl-Gorecka P, Müller CE et al (2017) P2Y2 receptor modulates shear stress-induced cell alignment and actin stress fibers in human umbilical vein endothelial cells. Cell Mol Life Sci 74:731–746. CrossRefPubMedGoogle Scholar
  18. 18.
    Weihs AM, Fuchs C, Teuschl AH et al (2014) Shock wave treatment enhances cell proliferation and improves wound healing by ATP release-coupled extracellular signal-regulated kinase (ERK) activation. J Biol Chem 289:27090–27104. CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Hoffmann C, Ziegler Reiner et al (2008) Agonist-selective, receptor-specific interaction of human P2Y receptors with beta-arrestin-1 and -2. J Biol Chem 283:30933–30941. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Knezevic L, Schaupper M, Mühleder S et al (2017) Engineering blood and lymphatic microvascular networks in fibrin matrices. Front Bioeng Biotechnol 5:1–12. CrossRefGoogle Scholar
  21. 21.
    Hackethal J, Mühleder S, Hofer A et al (2017) An effective method of Atelocollagen type 1/3 isolation from human placenta and its in vitro characterization in two-dimensional and three-dimensional cell culture applications. Tissue Eng Part C Methods 23:274–285. CrossRefPubMedGoogle Scholar
  22. 22.
    Rohringer S, Holnthoner W, Hackl M et al (2014) Molecular and cellular effects of in vitro shockwave treatment on lymphatic endothelial cells. PLoS One 9:e114806. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Sharov AA, Schlessinger D, Ko MSH (2015) ExAtlas: an interactive online tool for meta-analysis of gene expression data. J Bioinform Comput Biol 13:1550019. CrossRefPubMedPubMedCentralGoogle Scholar
  24. 24.
  25. 25.
    Liberzon A, Birger C, Thorvaldsdóttir H et al (2015) The molecular signatures database hallmark gene set collection. Cell Syst 1:417–425. CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Morpheus. Accessed 4 July 2019
  27. 27.
    Holnthoner W, Hohenegger K, Husa A-M et al (2015) Adipose-derived stem cells induce vascular tube formation of outgrowth endothelial cells in a fibrin matrix. J Tissue Eng Regen Med 9:127–136. CrossRefPubMedGoogle Scholar
  28. 28.
    Hasenberg T, Mühleder S, Dotzler A et al (2015) Emulating human microcapillaries in a multi-organ-chip platform. J Biotechnol 216:1–10. CrossRefPubMedGoogle Scholar
  29. 29.
    Wang L, Östberg O, Wihlborg AK et al (2003) Quantification of ADP and ATP receptor expression in human platelets. J Thromb Haemost 1:330–336. CrossRefPubMedGoogle Scholar
  30. 30.
    Sharma S, Rao A (2009) RNAi screening: tips and techniques. Nat Immunol 10:799–804. CrossRefPubMedPubMedCentralGoogle Scholar
  31. 31.
    Du D, Zhou Z, Zhu L et al (2018) TNF-α suppresses osteogenic differentiation of MSCs by accelerating P2Y2 receptor in estrogen-deficiency induced osteoporosis. Bone. CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Shinozaki Y, Shibata K, Yoshida K et al (2017) Transformation of astrocytes to a neuroprotective phenotype by microglia via P2Y1 Receptor downregulation. Cell Rep 19:1151–1164. CrossRefPubMedGoogle Scholar
  33. 33.
    Kobayashi K, Yamanaka H, Fukuoka T et al (2008) P2Y12 receptor upregulation in activated microglia is a gateway of p38 signaling and neuropathic pain. J Neurosci 28:2892–2902. CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Godecke S, Roderigo C, Rose CR et al (2012) Thrombin-induced ATP release from human umbilical vein endothelial cells. AJP Cell Physiol 302:C915–C923. CrossRefGoogle Scholar
  35. 35.
    Tian S, Quan H, Xie C et al (2011) YN968D1 is a novel and selective inhibitor of vascular endothelial growth factor receptor-2 tyrosine kinase with potent activity in vitro and in vivo. Cancer Sci 102:1374–1380. CrossRefPubMedGoogle Scholar
  36. 36.
    Savant S, La Porta S, Budnik A et al (2015) The orphan receptor Tie1 controls angiogenesis and vascular remodeling by differentially regulating Tie2 in Tip and stalk cells. Cell Rep 12:1761–1773. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    La Porta S, Roth L, Singhal M et al (2018) Endothelial Tie1-mediated angiogenesis and vascular abnormalization promote tumor progression and metastasis. J Clin Invest 128:834–845. CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Rafehi M, Burbiel JC, Attah IY et al (2017) Synthesis, characterization, and in vitro evaluation of the selective P2Y2 receptor antagonist AR-C118925. Purinergic Signal 13:89–103. CrossRefPubMedGoogle Scholar
  39. 39.
    Zhou Z, Chrifi I, Xu Y et al (2016) Uridine adenosine tetraphosphate acts as a proangiogenic factor in vitro through purinergic P2Y receptors. Am J Physiol Circ Physiol 311:H299–H309. CrossRefGoogle Scholar
  40. 40.
    Erb L, Weisman GA (2012) Coupling of P2Y receptors to G proteins and other signaling pathways. Wiley Interdiscip Rev Membr Transp Signal 1:789–803. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Andreev J, Galisteo ML, Kranenburg O et al (2001) Src and Pyk2 mediate G-protein-coupled receptor activation of epidermal growth factor receptor (EGFR) but are not required for coupling to the mitogen-activated protein (MAP) kinase signaling cascade. J Biol Chem 276:20130–20135. CrossRefPubMedGoogle Scholar
  42. 42.
    Strasser GA, Kaminker JS, Tessier-lavigne M, Dc W (2012) Microarray analysis of retinal endothelial tip cells identifies CXCR1 as a mediator of tip cell morphology and branching. Blood 115:5102–5110. CrossRefGoogle Scholar
  43. 43.
    Toro R, Prahst C, Mathivet T et al (2010) Identification and functional analysis of endothelial tip cell enriched genes. Blood 116:4025–4033. CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Siemerink MJ, Klaassen I, Vogels IMC et al (2012) CD34 marks angiogenic tip cells in human vascular endothelial cell cultures. Angiogenesis 15:151–163. CrossRefPubMedPubMedCentralGoogle Scholar
  45. 45.
    Felcht M, Luck R, Schering A et al (2012) Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling. J Clin Invest 122:1991–2005. CrossRefPubMedPubMedCentralGoogle Scholar
  46. 46.
    Potente M, Gerhardt H, Carmeliet P (2011) Basic and therapeutic aspects of angiogenesis. Cell 146:873–887. CrossRefGoogle Scholar
  47. 47.
    Benedito R, Rocha SF, Woeste M et al (2012) Notch-dependent VEGFR3 upregulation allows angiogenesis without VEGF-VEGFR2 signalling. Nature 484:110–114. CrossRefPubMedGoogle Scholar
  48. 48.
    Lampugnani MG, Orsenigo F, Gagliani MC et al (2006) Vascular endothelial cadherin controls VEGFR-2 internalization and signaling from intracellular compartments. J Cell Biol 174:593–604. CrossRefPubMedPubMedCentralGoogle Scholar
  49. 49.
    Khalafalla FG, Greene S, Khan H et al (2017) P2Y2 nucleotide receptor prompts human cardiac progenitor cell activation by modulating hippo signaling. Circ Res 121:1224–1236CrossRefPubMedPubMedCentralGoogle Scholar
  50. 50.
    Rocha SF, Schiller M, Jing D et al (2014) Esm1 modulates endothelial tip cell behavior and vascular permeability by enhancing VEGF bioavailability. Circ Res 115:581–590. CrossRefPubMedGoogle Scholar
  51. 51.
    Potente M, Mäkinen T (2017) Vascular heterogeneity and specialization in development and disease. Nat Rev Mol Cell Biol 18:477–494. CrossRefPubMedGoogle Scholar
  52. 52.
    Jakobsson L, Franco CA, Bentley K et al (2010) Endothelial cells dynamically compete for the tip cell position during angiogenic sprouting. Nat Cell Biol 12:943–953. CrossRefPubMedGoogle Scholar

Copyright information

© Springer Nature Switzerland AG 2019

Authors and Affiliations

  • Severin Mühleder
    • 1
    • 2
    • 3
  • Christiane Fuchs
    • 2
    • 4
    • 10
  • José Basílio
    • 5
  • Dorota Szwarc
    • 2
    • 4
  • Karoline Pill
    • 1
    • 2
  • Krystyna Labuda
    • 1
    • 2
  • Paul Slezak
    • 1
    • 2
  • Christian Siehs
    • 6
  • Johannes Pröll
    • 2
    • 7
    • 11
  • Eleni Priglinger
    • 1
    • 2
  • Carsten Hoffmann
    • 8
  • Wolfgang G. Junger
    • 1
    • 9
  • Heinz Redl
    • 1
    • 2
  • Wolfgang Holnthoner
    • 1
    • 2
    Email author
  1. 1.Ludwig Boltzmann Institute for Experimental and Clinical TraumatologyAUVA Research CenterViennaAustria
  2. 2.Austrian Cluster for Tissue RegenerationViennaAustria
  3. 3.Kompetenzzentrum für MechanoBiologie (INTERREG V-A AT-CZ ATCZ133)ViennaAustria
  4. 4.Department Life Science EngineeringUniversity of Applied Sciences Technikum WienViennaAustria
  5. 5.Department of Vascular Biology and Thrombosis ResearchMedical University of ViennaViennaAustria
  6. 6.Mag. Dipl.-Ing. Dr. Christian Siehs, IT-Services, GLN 9110002040261ViennaAustria
  7. 7.Center for Medical ResearchJohannes Kepler UniversityLinzAustria
  8. 8.Institut für Molekulare Zellbiologie, CMB-Center for Molecular BiomedicineUniversitätsklinikum Jena, Friedrich-Schiller-UniversitätJenaGermany
  9. 9.Department of SurgeryBeth Israel Deaconess Medical Center, Harvard Medical SchoolBostonUSA
  10. 10.Wellman Center for PhotomedicineMassachusetts General HospitalBostonUSA
  11. 11.Red Cross Blood Transfusion ServiceLinzAustria

Personalised recommendations