Abstract
Leukocyte recruitment is a fundamental event in the response of the innate immune system to injury. This process is promoted in part by the opening of endothelial cell adherens junctions that allows leukocyte extravasation through gaps between adjacent endothelial cells. VE-cadherin is a key component of endothelial cell adherens junctions and a negative regulator of leukocyte emigration. Accumulating evidence implicates bone morphogenetic protein (BMP) 4 as a critical regulator in vascular biology, but its role in leukocyte extravasation in vitro and in vivo has not been investigated so far. To assess the impact of BMP4 on leukocyte emigration in vivo, we used the thioglycollate-induced peritonitis model. C57BL/6 mice were intraperitoneally (i.p.) injected with recombinant BMP4 in addition to thioglycollate. Compared to solvent-treated controls, we observed higher accumulation of leukocytes in the peritoneal lavage of BMP4-treated mice indicating that BMP4 promotes leukocyte diapedesis into the inflamed peritoneal cavity. Endothelial cell-specific deletion of BMP4 in mice markedly diminished leukocyte diapedesis following thioglycollate administration suggesting that endothelial BMP4 is required for leukocyte recruitment. Consistent with these in vivo results, transwell migration assays with human umbilical vein endothelial cells (HUVECs) in vitro revealed that recombinant BMP4 enhanced leukocyte transmigration through the endothelial monolayer. Conversely, silencing of endothelial BMP4 by siRNA dampened leukocyte diapedesis in vitro. Mechanistic studies showed that loss of BMP4 improved endothelial junction stability by upregulation of VE-cadherin expression in vitro and in vivo. Vice versa, treatment of HUVECs with recombinant BMP4 decreased expression of VE-cadherin and impaired endothelial junction stability shown by Western blotting and immunocytochemistry. Finally, severe endothelial damage in HUVECs in response to serum of patients collected 24 h after survived cardiac arrest was accompanied by increase in leukocyte migration in transwell assays and activation of the BMP pathway most probably by upregulation of endothelial BMP4 RNA and protein expression. Collectively, the present study provides novel evidence that endothelial BMP4 controls leukocyte recruitment through a VE-cadherin-dependent mechanism and that BMP4-induced inflammation might be involved in the pathogenesis of endothelial cell damage following successful resuscitation after cardiac arrest.
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Acknowledgements
We are indebted to Adrian Bauer and Jessica Beckert for their outstanding technical assistance.
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T.H. designed and performed the research, analyzed the data, and wrote the paper; L.A., G.W., E.H., V.G., and A.H. performed the research and analyzed the data; J.S.E., P.D., and S.G. analyzed the data and contributed analytical tools; K.F. and H.J.B. provided blood samples; C.B. and M.M. wrote the paper.
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All procedures performed in studies involving murine or human tissues were in accordance with the ethical standards of the institution and/or national research.
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The authors declare that they have no conflict of interest.
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This work was supported by Deutsche Forschungsgemeinschaft to T.H. (HE 7432/1-1) and M.M. (MO 973/8-1) and by MSD to T.H.
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BMP4 expression in primary microvascular endothelial cells isolated from EC-BMP4+/+ and EC-BMP4−/− mice. Cells were lysed and proteins were extracted to perform Western blot analysis with a BMP4 antibody and α-tubulin antibody. (PDF 585 kb)
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Helbing, T., Arnold, L., Wiltgen, G. et al. Endothelial BMP4 Regulates Leukocyte Diapedesis and Promotes Inflammation. Inflammation 40, 1862–1874 (2017). https://doi.org/10.1007/s10753-017-0627-0
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DOI: https://doi.org/10.1007/s10753-017-0627-0