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Bone Morphogenetic Protein-Modulator BMPER Regulates Endothelial Barrier Function

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Abstract

The endothelium serves as a selective barrier and controls the exchange of nutrients, hormones, and leukocytes between blood and tissues. Molecular mechanisms contributing to the pathogenesis of endothelial barrier dysfunction remain incompletely understood. Accumulating evidence implicates bone morphogenetic protein (BMP)-modulator BMPER as a key regulator in endothelial biology. Herein, we analyze the impact of BMPER in the control of endothelial barrier function. To assess the role of BMPER in vascular barrier function in mice, we measured the leakage of Evans blue dye from blood into interstitial lung tissue. BMPER+/− mice exhibited a significantly higher degree of vascular leak compared with wild-type siblings. In accordance with our in vivo observation, siRNA-based BMPER knockdown in human umbilical endothelial cells increased endothelial permeability measured by FITC-dextran passage in transwell assays. Mechanistically, BMPER knockdown reduced the expression of VE-cadherin, a pivotal component of endothelial adherens junctions. Conversely, recombinant human BMPER protein upregulated VE-cadherin protein levels and improved endothelial barrier function in transwell assays. The effects of BMPER knockdown on VE-cadherin expression and endothelial permeability were induced by enhanced BMP activity. Supporting this notion, activation of BMP4-Smad-Id1 signaling reduced VE-cadherin levels and impaired endothelial barrier function in vitro. In vivo, Evans blue dye accumulation was higher in the lungs of BMP4-treated C57BL/6 mice compared to controls indicating that BMP4 increased vascular permeability. High levels of BMPER antagonized BMP4-Smad5-Id1 signaling and prevented BMP4-induced downregulation of VE-cadherin and endothelial leakage, suggesting that BMPER exerts anti-BMP effects and restores endothelial barrier function. Taken together, this data demonstrates that BMPER-modulated BMP pathway activity regulates VE-cadherin expression and vascular barrier function.

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Acknowledgments

We are indebted to Bianca Engert for her outstanding technical assistance.

Authors’ Contributions

T.H. designed and performed the research, analyzed the data, and wrote the paper; G.W., A.H., E.K., L.A., and A.B. performed the research and analyzed the data; K.F. and P.D. performed the research; S.G. and J.S.E. analyzed the data and contributed analytical tools; C.P. and C.B. wrote the paper; and M.M. designed the research and wrote the paper.

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Authors and Affiliations

  1. Department of Cardiology and Angiology, Heart Center Freiburg University, Hugstetter Strasse 55, 79106, Freiburg im Breisgau, Germany

    Thomas Helbing, Gwendoline Wiltgen, Alexandra Hornstein, Elena Z. Brauers, Linus Arnold, Adrian Bauer, Jennifer S. Esser, Philipp Diehl, Sebastian Grundmann, Christoph Bode & Martin Moser

  2. Faculty of Medicine, University of Freiburg, Freiburg im Breisgau, Germany

    Thomas Helbing, Gwendoline Wiltgen, Alexandra Hornstein, Elena Z. Brauers, Linus Arnold, Adrian Bauer, Jennifer S. Esser, Philipp Diehl, Sebastian Grundmann, Katrin Fink, Christoph Bode & Martin Moser

  3. Department of Emergency Medicine, University Hospital of Freiburg, Freiburg im Breisgau, Germany

    Katrin Fink

  4. McAllister Heart Institute, University of North Carolina at Chapel Hill, Chapel Hill, NC, 27599, USA

    Cam Patterson

  5. New York-Presbyterian Hospital, New York City, NY, 10065, USA

    Cam Patterson

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  1. Thomas Helbing
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Corresponding author

Correspondence to Thomas Helbing.

Ethics declarations

All procedures performed in studies involving murine or human tissues were in accordance with the ethical standards of the institution and/or national research committee and with the 1964 Helsinki declaration and its later amendments. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

Conflict of Interest

The authors declare that they have no conflict of interest.

Funding

This work was supported by Deutsche Forschungsgemeinschaft Mo973/6-1 to M.M. and by intramural funds to T.H. and M.M.

ELECTRONIC SUPPLEMENTARY MATERIAL

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Supplement 1

(A) Time course of Smad1/5 phosphorylation in response to BMPER. HUVEC were incubated to BMPER (right panel, 50 ng/ml) for indicated periods. Total cells lysates were used for western blotting with an anti p-Smad1/5 antibody. B-tubulin served as a loading control. (* = P < 0.05, n = 4) (B) Smad5 dependent gene transcription was not altered by increasing BMPER concentrations. Smad5 luciferase reporter plasmid (3GC2wt-luc) was transiently co-transfected with pCMV-β-gal in HEK293 cells. After stimulation with BMPER (250 ng/ml) for additional 20 h luciferase and β-galactosidase activity were measured. Luciferase activity was normalized to β-galactosidase activity and is expressed as mean +/− SD. (* = P < 0.05 versus control, # = P < 0.05 versus BMP4, n = 4) (C) BMPER knockdown does not lead to changes in occludin expression. HUVEC were transfected with a scrsiRNA or BMPER siRNA (siBMPERI or II) for 48 h. Cell extracts were analyzed by western blot for occludin, VE-cadherin and α-tubulin. Reduction of VE-cadherin after transfection of BMPER siRNA served as a positive control. (JPG 1001 kb)

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Helbing, T., Wiltgen, G., Hornstein, A. et al. Bone Morphogenetic Protein-Modulator BMPER Regulates Endothelial Barrier Function. Inflammation 40, 442–453 (2017). https://doi.org/10.1007/s10753-016-0490-4

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