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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

Abstract

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.

Keywords

Endothelial Purinergic Angiogenesis P2Y2 Tip cell Sprouting 

Notes

Acknowledgements

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

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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

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