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Endothelial BMP4 Promotes Leukocyte Rolling and Adhesion and Is Elevated in Patients After Survived Out-of-Hospital Cardiac Arrest

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Abstract

Leukocyte recruitment is a fundamental step in the inflammatory response during ischemia/reperfusion injury (IRI). Rolling and adhesion of leukocytes to activated endothelium promote tissue inflammation after IRI and require presentation of adhesion molecules E-selectin and ICAM-1 on the endothelial surface. Bone morphogenetic protein (BMP) 4 is a prominent member of the BMP family expressed and secreted by endothelial cells. BMP4 derived from endothelial cells has important functions in vascular disease but its influence on the leukocyte adhesion cascade during inflammation is incompletely understood. In the present study, we challenged mice with an inducible endothelial-specific BMP4 deletion (referred to as EC-BMP4−/− mice) and their control littermates (EC-BMP4+/+) with thioglycollate i.p. and assessed extravasation of different leukocyte subsets during peritonitis. Peritoneal lavages were performed and peritoneal cells were counted. Total cell count in lavages of EC-BMP4−/− mice was markedly reduced compared with lavages of EC-BMP4+/+ mice. FACS analyses of thioglycollate-elicited peritoneal cells revealed that diverse leukocyte subsets were reduced in EC-BMP4−/− mice. Intravital microscopy of cremaster venules demonstrated that rolling and adhesion of leukocytes were significantly diminished in EC-BMP4−/− mice in comparison with control mice in response to TNFα. These observations indicate that endothelial BMP4 is essential for rolling, adhesion, and extravasation of leukocytes in vivo. To understand the underlying mechanisms, levels of endothelial adhesion molecules E-selectin and ICAM-1 were quantified in EC-BMP4−/− and EC-BMP4+/+ mice by quantitative PCR and Western blotting. Interestingly, ICAM-1 and E-selectin expressions were reduced in the hearts of EC-BMP4−/− mice. Next we confirmed pro-inflammatory properties of BMP4 in a gain of function experiments and found that administration of recombinant BMP4 in male C57BL/6 mice increased leukocyte rolling and adhesion in cremaster venules in vivo. To assess the regulation of BMP4 in inflammatory disease in humans, we collected plasma samples of patients from day 0 to day 7 after survived out-of-hospital cardiac arrest (OHCA, n = 42). Remarkably, plasma of OHCA patients contained significantly higher BMP4 protein levels compared with patients with coronary artery disease (CAD, n = 12) or healthy volunteers (n = 11). Subgroup analysis revealed that elevated plasma BMP4 levels after ROSC are associated with decreased survival and unfavorable neurological outcome. Collectively, endothelial BMP4 is a potent activator of inflammation in vivo that promotes rolling, adhesion, and extravasation of leukocyte subsets by induction of E-selectin and ICAM-1. Elevation of plasma BMP4 levels in the post-resuscitation period suggests that BMP4 contributes to pathophysiology and poor outcome of post-cardiac arrest syndrome.

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Acknowledgments

We are indebted to Jessica Beckert for her outstanding technical assistance.

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Correspondence to Thomas Helbing.

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All animal procedures were approved by the local ethics committee (Regierungspräsidium Freiburg) and were performed according to the respective guidelines. The study was approved by the ethics committee of the University of Freiburg and confirms to the tenets of the declaration of Helsinki.

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Electronic Supplementary Material

Supplement 1

Flow cytometric analysis of mouse thioglycollate-elicited peritoneal cells. Gating strategy used to identify different subsets of peritoneal cells of Figure 1. (AI 1441 kb)

Supplement 2

BMP4 protein expression in total heart tissue extracted from EC-BMP4+/+ and EC-BMP4−/− mice. Cells were lysed and proteins were extracted to perform western blot analysis with an anti-BMP4 and anti-α-tubulin antibody. (AI 527 kb)

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Arnold, L., Weberbauer, M., Herkel, M. et al. Endothelial BMP4 Promotes Leukocyte Rolling and Adhesion and Is Elevated in Patients After Survived Out-of-Hospital Cardiac Arrest. Inflammation 43, 2379–2391 (2020). https://doi.org/10.1007/s10753-020-01307-9

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