Signals of the Neuropilin-1–MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells

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Signals of the Neuropilin-1–MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells Signals of the Neuropilin-1−MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells This information is current as of September 27, 2021. Maryam Rezaei, Ana C. Martins Cavaco, Jochen Seebach, Stephan Niland, Jana Zimmermann, Eva-Maria Hanschmann, Rupert Hallmann, Hermann Schillers and Johannes A. Eble J Immunol published online 28 January 2019 Downloaded from http://www.jimmunol.org/content/early/2019/01/27/jimmun ol.1801346 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2019/01/27/jimmunol.180134 Material 6.DCSupplemental Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists by guest on September 27, 2021 • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2019 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published January 28, 2019, doi:10.4049/jimmunol.1801346 The Journal of Immunology Signals of the Neuropilin-1–MET Axis and Cues of Mechanical Force Exertion Converge to Elicit Inflammatory Activation in Coherent Endothelial Cells Maryam Rezaei,* Ana C. Martins Cavaco,* Jochen Seebach,† Stephan Niland,* Jana Zimmermann,* Eva-Maria Hanschmann,‡ Rupert Hallmann,* Hermann Schillers,x and Johannes A. Eble* The neuropilin-1 (NRP1)-MET signaling axis regulates the motility of individual endothelial cells (ECs). It is unknown how this signaling pathway affects the endothelial barrier in coherent ECs forming a tight monolayer. We hypothesized that it is involved both in modulation of the endothelial barrier and in EC activation. To investigate the role of NRP1–MET signaling in inflammatory processes (e.g., systemic inflammatory response syndrome [SIRS] or snakebite-induced SIRS-like conditions), we employed the Downloaded from C-type lectin-related protein rhodocetin-ab (RCab) as a specific trigger of this signal axis in ECs in vitro. In coherent HUVECs, RCab reinforced the actin cytoskeleton and increased cell stiffness, thus favoring vascular endothelial cadherin–mediated trans- mission of intercellular forces. Increased cell stiffness was associated with enhanced activation of RhoA and nuclear translocation of NF-kB. Simultaneously, RCab-triggered signaling via the NRP1–MET axis increased EC monolayer permeability, induced transcription of proinflammatory genes such as ICAM-1 and, consequently, leukocyte tethering. The RCab-induced transcriptome differed from that induced by hepatocyte growth factor, although in both cases the same tyrosine kinase, MET, was involved. This http://www.jimmunol.org/ was due to RCab-mediated recruitment of the MET coreceptor NRP1 and additional Rho-mediated activation of the actomyosin system. RCab induced similar transcriptional and cellular changes if external shear forces were applied. These data highlight the modulatory role of NRP1 as MET coreceptor, and they explain how some snake venoms induce SIRS-like conditions. Addi- tionally, this study demonstrates that inflammatory activation of coherent ECs is triggered by converging signals that are induced by NRP1–MET signaling and influenced by intercellular forces. The Journal of Immunology, 2019, 202: 000–000. he inner surface of blood vessels is lined by a monolayer The interaction of leukocytes with ECs during TEM is a mul- of endothelial cells (ECs). Tightly connected to each tistep procedure, involving exposure of leukocyte-adhesive pro- by guest on September 27, 2021 T other, they form a barrier between the blood compartment teins, such as ICAM-1, on the luminal surface of ECs (5, 6). Being and surrounding vascular and stromal cells (1). Moreover, this EC a marker of this EC activation, ICAM-1 expression is regulated by monolayer controls the transport of biochemical compounds and various inflammatory cytokines (5, 7). Characteristically activated of cells between bloodstream and vessel-surrounding stroma. during inflammation (8), NF-kB is a transcription factor that Leukocytes cross the monolayer of tightly interconnected ECs regulates transcription of the ICAM-1 gene (5, 7). To fulfill their during immune surveillance and inflammatory defense (2). Dys- functions, ICAM-1 molecules, which are stored in intracellular function of this barrier increases vessel permeability and leakage vesicles, have to be exposed on the cell surface. Moreover, for and disturbs transendothelial migration (TEM) of leukocytes. This leukocyte attachment to ICAM-1–exposing ECs, the ICAM-1 becomes manifest in edema formation and malfunctioning of the molecules have to be arrayed in a certain pattern on the cell immune defense, such as in chronic inflammatory diseases. A surface, a process that depends on the stiffness of the underlying local inflammatory response may become life-threatening if it matrix (9). reaches out to the entire vasculature, resulting in systemic inflam- ECs need to withstand mechanical shear forces induced by the matory response symptoms/sepsis (3, 4). bloodstream. They are able to do this because of their actomyosin *Institute of Physiological Chemistry and Pathobiochemistry, Westfalian Wilhelm Uni- The microarray data presented in this article have been submitted to the Gene Ex- versity Mu¨nster, 48149 Mu¨nster, Germany; †Institute of Anatomy and Vascular Biology, pression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/) under acces- Westfalian Wilhelm University Mu¨nster, 48149 Mu¨nster, Germany; ‡Department of sion number GSE121297. Neurology, Medical Faculty, Heinrich-Heine University Du¨sseldorf, 40225 Du¨sseldorf, x Address correspondence and reprint requests to Dr. Johannes A. Eble, Institute of Germany; and Institute of Physiology II, Westfalian Wilhelm University Mu¨nster, 48149 Physiological Chemistry and Pathobiochemistry, University of Mu¨nster, Waldeyerstraße15, Mu¨nster, Germany 48149 Mu¨nster, Germany. E-mail address: [email protected] ORCIDs: 0000-0002-2055-8656 (S.N.); 0000-0003-3288-1706 (R.H.); 0000-0001- The online version of this article contains supplemental material. 6090-4771 (H.S.); 0000-0001-9156-2137 (J.A.E.). Abbreviations used in this article: AFM, atomic force microscopy; CBT, Cell Border Received for publication October 9, 2018. Accepted for publication December 27, Tracker; EC, endothelial cell; ECM, extracellular matrix; HGF, hepatocyte growth 2018. factor; NRP1, neuropilin-1; pfp, percentage of false predictions; PKB/Akt, protein This work was supported by Deutsche Forschungsgemeinschaft (DFG) Grant kinase B; RCab, rhodocetin-ab; ROCK, Rho-associated kinase; siRNA, small inter- SFB1009 A09 (to J.A.E.) and Wilhelm Sander-Stiftung Grant 2016.113.1 (to J.A.E.). fering RNA; SIRS, systemic inflammatory response syndrome; TEM, transendothe- As a member of the DFG-funded Cells-in-Motion Cluster of Excellence, J.A.E. also lial migration; TER, transendothelial electrical resistance; VE-cadherin, vascular received financial support from there (EXC1003-CiM). J.S. received funding from endothelial cadherin; VEGF, vascular endothelial growth factor. the Cells-in-Motion Cluster of Excellence (FF-2016-15). Copyright Ó 2019 by The American Association of Immunologists, Inc. 0022-1767/19/$37.50 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1801346 2 NRP1–MET SIGNALS AND CUES FROM INTERCELLULAR FORCES CONVERGE system and their anchoring proteins, which connect ECs to the thereby promoting cell migration (29). In addition, in an in vivo underlying extracellular matrix (ECM) and neighboring cells tumor model, RCab selectively disrupted blood vessels of the (10, 11). The actomyosin machinery is a complex network of tumor but not of healthy tissues (31). F-actin bundles, actin-organizing proteins, such as members of the To better understand these surprising findings, the effect of Rho family, and motor proteins (12, 13). Mechanical forces be- RCab on monolayers of confluent ECs was investigated in an tween ECs and their surrounding are transmitted via transmem- in vitro system at transcriptional, protein-chemical, and cellular brane proteins such as integrins and cadherins. These proteins level. anchor an EC to the ECM and neighboring cells, respectively. Integrins are heterodimeric proteins that bind to ECM proteins and are connected to the actin cytoskeleton via adapter proteins (14). Materials and Methods They are part of adhesomes, supramolecular complexes in which Cell culture and silencing of NRP1 and MET several adapter and signaling proteins help integrins to transmit HUVECs (Promocell, Heidelberg, Germany) were cultivated, unless mechanical forces between a cell and its ECM (15). Two main otherwise stated, up to passage 5 on rat tail tendon collagen-I (Corning, types of adhesomes are distinguished: focal complexes and focal Tewksbury, MA) in complete EC growth medium (Promocell) supple- mented with 2% heat-inactivated FBS and 1% penicillin/streptomycin adhesions. The latter can bear higher mechanical forces and (Invitrogen Life Technologies).
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