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Angiogenesis

, Volume 17, Issue 3, pp 587–601 | Cite as

Regulation of endothelial signaling and migration by v-ATPase

  • Sebastian Rath
  • Johanna Liebl
  • Robert Fürst
  • Angelika M. Vollmar
  • Stefan ZahlerEmail author
Original Paper

Abstract

The vacuolar ATPase (v-ATPase) is a proton pump, able to acidify intracellular compartments and the pericellular space. v-ATPase has extensively been studied in various functional contexts, e.g., migration of tumor cells, and inhibition of v-ATPase has been proven as intriguing novel therapeutic concept. Since the role of v-ATPase in endothelial cell migration and angiogenesis has scarcely been investigated, we examined the consequences of pharmacological inhibition of v-ATPase (by concanamycin) on proliferation, migration, VEGF-receptor 2 (VEGFR2) trafficking and signaling, as well as Notch-mediated transcription in endothelial cells [human microvascular endothelial cells (HMEC-1) and human umbilical vein endothelial cells (HUVEC)] Treatment of the cells with 3 or 10 nM of the v-ATPase inhibitor concanamycin for 48 h or longer inhibited proliferation and arrested cell cycle in the G2/M phase in HMEC-1, while a G1 phase arrest occurred in HUVEC. Already after 24 h these concentrations reduced migration (scratch assay, chemotactic gradient). Activation of the small GTPase Rac1 in freshly adherent cells was reduced by concanamycin. Downstream signaling of the VEGFR2 (phosphorylation of ERK1/2 and AKT), as well as autophosphorylation of VEGFR2 were inhibited. VEGFR2 on the cell surface was reduced, and sequestered in a lysosomal compartment. In addition, concanamycin blocked transcription of the Notch target genes Hey1 and Hey2 after stimulation with DLL4. Since the impaired signaling pathways (Rac-1, VEGFR2, Notch) all depend on vesicular recycling circuits, we conclude that the disturbance of these is the main mode of action of v-ATPase inhibition in endothelial cells, offering an attractive multi-factorial anti-angiogenic approach.

Keywords

Angiogenesis v-ATPase HUVEC Endothelial Concanamycin 

Notes

Acknowledgments

The technical assistance by Jana Peliskova and Bianca Hager is gratefully acknowledged. The work was partly supported by the Deutsche Forschungsgemeinschaft (DFG), FOR 1406, project VO376-14/15 and FU691-9/1.

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Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Sebastian Rath
    • 1
  • Johanna Liebl
    • 1
  • Robert Fürst
    • 1
    • 2
  • Angelika M. Vollmar
    • 1
  • Stefan Zahler
    • 1
    Email author
  1. 1.Department of PharmacyLudwig-Maximilians-Universität MünchenMunichGermany
  2. 2.Institute of Pharmaceutical Biology, BiocenterGoethe-University FrankfurtFrankfurtGermany

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