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Regulation of endothelial signaling and migration by v-ATPase

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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.

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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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Authors and Affiliations

  1. Department of Pharmacy, Ludwig-Maximilians-Universität München, Butenandtstr. 5-13, 81377, Munich, Germany

    Sebastian Rath, Johanna Liebl, Robert Fürst, Angelika M. Vollmar & Stefan Zahler

  2. Institute of Pharmaceutical Biology, Biocenter, Goethe-University Frankfurt, Max-von-Laue-Str. 9, 60438, Frankfurt, Germany

    Robert Fürst

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  1. Sebastian Rath
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  2. Johanna Liebl
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Correspondence to Stefan Zahler.

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Rath, S., Liebl, J., Fürst, R. et al. Regulation of endothelial signaling and migration by v-ATPase. Angiogenesis 17, 587–601 (2014). https://doi.org/10.1007/s10456-013-9408-z

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