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Sprouty2 expression controls endothelial monolayer integrity and quiescence

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Abstract

Vascular integrity is fundamental to the formation of mature blood vessels and depends on a functional, quiescent endothelial monolayer. However, how endothelial cells enter and maintain quiescence in the presence of angiogenic factors is still poorly understood. Here we identify the fibroblast growth factor (FGF) antagonist Sprouty2 (Spry2) as a key player in mediating endothelial quiescence and barrier integrity in mouse aortic endothelial cells (MAECs): Spry2 knockout MAECs show spindle-like shapes and are incapable of forming a functional, impermeable endothelial monolayer in the presence of FGF2. Whereas dense wild type cells exhibit contact inhibition and stop to proliferate, Spry2 knockout MAECs remain responsive to FGF2 and continue to proliferate even at high cell densities. Importantly, the anti-proliferative effect of Spry2 is absent in sparsely plated cells. This cell density-dependent Spry2 function correlates with highly increased Spry2 expression in confluent wild type MAECs. Spry2 protein expression is barely detectable in single cells but steadily increases in cells growing to high cell densities, with hypoxia being one contributing factor. At confluence, Spry2 expression correlates with intact cell–cell contacts, whereas disruption of cell–cell contacts by EGTA, TNFα and thrombin decreases Spry2 protein expression. In confluent cells, high Spry2 levels correlate with decreased extracellular signal-regulated kinase 1/2 (Erk1/2) phosphorylation. In contrast, dense Spry2 knockout MAECs exhibit enhanced signaling by Erk1/2. Moreover, inhibiting Erk1/2 activity in Spry2 knockout cells restores wild type cobblestone monolayer morphology. This study thus reveals a novel Spry2 function, which mediates endothelial contact inhibition and barrier integrity.

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Acknowledgments

We thank O. Sansom and I. Ahmad, who kindly provided the Floxed-Spry2 mouse strains (Beatson Institute, Glasgow, Scotland). We are grateful to M. A. Cabrita and I. Bhattacharya for helpful discussions. We thank Sigrid Strom, Ina Kalus and Elvira Haas for critical review of the manuscript. This work was supported by grants from the Swiss National Science Foundation to E. J. B. and from the University of Zürich.

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The authors declare no conflict of interest.

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Authors and Affiliations

  1. Division of Internal Medicine, University Hospital Zurich, Gloriastrasse 30, GLO30 J14, 8091, Zurich, Switzerland

    Martin Peier, Thomas Walpen, Edouard Battegay & Rok Humar

  2. Department of Biomedicine, University of Basel, 4058, Basel, Switzerland

    Gerhard Christofori

  3. Zurich Center for Integrative Human Physiology (ZIHP), Rämistrasse 100, 8091, Zurich, Switzerland

    Edouard Battegay & Rok Humar

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Correspondence to Rok Humar.

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10456_2012_9330_MOESM1_ESM.tif

Supplementary figure 1 Male and female MAECs show similar FGF2-Erk1/2 signaling and density dependent Spry2 expression (A) Confluent female (♀) and male (♂) MAECs were starved and stimulated with FGF2 for the indicated time. The cells were then lysed and analyzed for ERK1/2 phosphorylation and total Erk1/2 expression. Relative phosphorylation levels of Erk1/2 were quantified by densitometry. Bars show values as fold change of unstimulated cells ± s.e.m. n = 3. (B) Basal Spry2 expression and Erk1/2 activity in sparse and confluent female and male MAECs. Cells were plated at sparse (3,000 cells/cm2) or confluent conditions (50,000 cells/cm2), starved, lysed and analyzed by immunoblotting (TIFF 6264 kb)

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Peier, M., Walpen, T., Christofori, G. et al. Sprouty2 expression controls endothelial monolayer integrity and quiescence. Angiogenesis 16, 455–468 (2013). https://doi.org/10.1007/s10456-012-9330-9

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