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Purinergic P2Y2 receptors modulate endothelial sprouting

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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.

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Data and materials availability

Further information and requests for materials should be directed to the corresponding author Wolfgang Holnthoner. All raw files from mRNA analyses are available in the gene expression omnibus (GEO) under accession number GSE133795.

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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.

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Author notes

  1. Christiane Fuchs

    Present address: Wellman Center for Photomedicine, Massachusetts General Hospital, Boston, MA, USA

Authors and Affiliations

  1. Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, AUVA Research Center, Donaueschingenstrasse 13, 1200, Vienna, Austria

    Severin Mühleder, Karoline Pill, Krystyna Labuda, Paul Slezak, Eleni Priglinger, Wolfgang G. Junger, Heinz Redl & Wolfgang Holnthoner

  2. Austrian Cluster for Tissue Regeneration, Vienna, Austria

    Severin Mühleder, Christiane Fuchs, Dorota Szwarc, Karoline Pill, Krystyna Labuda, Paul Slezak, Johannes Pröll, Eleni Priglinger, Heinz Redl & Wolfgang Holnthoner

  3. Kompetenzzentrum für MechanoBiologie (INTERREG V-A AT-CZ ATCZ133), Vienna, Austria

    Severin Mühleder

  4. Department Life Science Engineering, University of Applied Sciences Technikum Wien, Vienna, Austria

    Christiane Fuchs & Dorota Szwarc

  5. Department of Vascular Biology and Thrombosis Research, Medical University of Vienna, Vienna, Austria

    José Basílio

  6. Mag. Dipl.-Ing. Dr. Christian Siehs, IT-Services, GLN 9110002040261, Vienna, Austria

    Christian Siehs

  7. Center for Medical Research, Johannes Kepler University, Linz, Austria

    Johannes Pröll

  8. Institut für Molekulare Zellbiologie, CMB-Center for Molecular Biomedicine, Universitätsklinikum Jena, Friedrich-Schiller-Universität, Jena, Germany

    Carsten Hoffmann

  9. Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, 02215, MA, USA

    Wolfgang G. Junger

  10. Red Cross Blood Transfusion Service, Linz, Austria

    Johannes Pröll

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  1. Severin Mühleder
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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.

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Correspondence to Wolfgang Holnthoner.

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Supplementary material 1 (DOCX 1051 kb)

18_2019_3213_MOESM2_ESM.avi

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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Mühleder, S., Fuchs, C., Basílio, J. et al. Purinergic P2Y2 receptors modulate endothelial sprouting. Cell. Mol. Life Sci. 77, 885–901 (2020). https://doi.org/10.1007/s00018-019-03213-2

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