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. 2020 Feb 4;10:1778. doi: 10.1038/s41598-020-58374-z

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© The Author(s) 2020

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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P. gingivalis promotes EMV shedding and alters endothelial cell viability. (A) The generation of EMV from naïve EC (control) and following 24 h of infection with P. gingivalis (Pg) (MOI = 100) or stimulation with Pg-LPS (1μg/ml) was measured in the supernatant by prothrombinase assay. Concentrations are represented by mean +/− SD from 3 independent experiments; *p < 0.05 vs control (unstimulated EC). (B) Metabolic activity of EC infected with Pg (MOI = 100) or exposed to EMV_Ctrl (upper panel) and EMV_Pg (lower panel) (5, 10, 20 and 30 nM) for 24 h measured by AlamarBlue assay. Results are expressed as mean +/− SD from 3 independent experiments; *p < 0.05 vs control (unstimulated EC). (C) Live-Dead assay to evaluate the ratio of live EC versus dead EC in cells exposed to P. gingivalis (Pg) (MOI:100) and EMV_Pg (5, 10, 20 and 30 nM) for 24 h. Results are expressed as percentage of live and dead EC (mean +/−SD). (D) Immunofluorescence imaging of live-dead staining (green: live EC, red: dead EC) for each condition after 24 hours of exposure. (E) Evaluation of type of cell death by flow cytometry after P. gingivalis infection and EMV_Pg (20 and 30 nM) exposure for 24 h. EC were labelled with Annexin-V FITC and propidium iodide (IPPE). All data were expressed as mean ± SD. *(p < 0.05 vs untreated cells).