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. 2022 Mar 21;13:1521. doi: 10.1038/s41467-022-29233-4

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© The Author(s) 2022

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 5 — a Transient elastase-treatment-induced eATP release from EC cultures is significantly mitigated by PBN treatment at 6- and 12 h. *P < 0.0001 vs. elastase; n = 12/group. b Elastase- and/or cytomix-induced eATP release by ECs is attenuated by Panx1 inhibition via PBN treatment compared with untreated controls. * P < 0.0001 vs. control; ^#P = 0.0009 vs. elastase; ^δP < 0.0001 vs. cytomix; n = 12/group. c Schematic showing the in vitro transwell model to demonstrate transendothelial migration of polymorphonuclear neutrophils (PMNs). Fluorescent-labeled primary murine-derived PMNs were included in the top chamber containing ECs and cocultured for 24 h. d Exposure to elastase or cytomix showed a significant increase in neutrophil transmigration, which was significantly attenuated by pretreatment with PBN. *P < 0.0001 vs. control; ^#P = 0.0002 vs. elastase; ^δP < 0.0001 vs. cytomix; n = 12/group. e Schematic depicting conditioned media transfer (CMT)-based experiments from elastase-treated ECs to macrophages with/without pretreatment with inhibitors. f, g CMT from elastase-treated ECs to macrophages induces a significant upregulation of IL-1β and HMGB1 secretion, which was blocked by pretreatment of ECs with apyrase or PBN, as well as pretreatment of macrophages with P2X7 inhibitor (A80). ^δP < 0.0001 vs. EC → MФ; *P = 0.0001, **P = 0.0009, ^#P = 0.0005 and ^##P = 0.03 vs. EC^elastase → MФ; n = 8/group. All comparative data above are represented as mean values ± SEM and statistical analyses were done by one-way ANOVA. h Panx1-dependent ATP release from ECs stimulates mtDNA release that is prevented by PBN and P2X7R inhibition. CMT from elastase-induced ECs was performed on macrophages. DNA was isolated from cytosolic fractions of macrophages and the levels of mitochondrial (mt)DNA were analyzed by quantitative RT-PCR. CMT from elastase-treated ECs to macrophages induces a significant upregulation of mtDNA release, which was blocked by pretreatment of ECs with apyrase or PBN, as well as pretreatment of macrophages with P2X7 inhibitor. *P = 0.0005; **P = 0.02, ^#P = 0.01, and ^δP = 0.004 vs. EC^elastase → MФ; n = 8/group. Data are presented as fold change relative to EC → MФ and compared using two-tailed Wilcoxon test. i Schematic displaying the crosstalk between ECs and macrophages via Panx1-dependent release from elastase-induced ECs that stimulates P2X7R and mtDNA release from macrophages.