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. 2024 Feb 12;22:60. doi: 10.1186/s12951-024-02304-y

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

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Fig. 4 — Functional effects of MSC-EVs on ECs in vitro. A Cellular up-take of MSC-EVs by untreated (control) or IL-1β and TNF-α pre-incubated MCECs by fluorescent microscopy. MSC-EVs were fluorescently labelled with DIO (green) and incubated with cells for 2, 4 or 24 h. (Left) Representative images of MCECs incubated with MSC-EVs. (Right) Mean fluorescence intensity (MFI) of MCECs incubated with MSC-EVs measured at different time points and analyzed by ImageJ software. Scale bar = 100 µm. B Expression of selected endothelial cell activation markers (CD54, CD106, CD62E, CD62P) on MCECs: untreated cells (Control), pre-incubated with IL-1β, TNF-α and/or treated with MSC-EVs for 4 h or 24 h, measured by flow cytometry. C (Left) Proangiogenic potential of MCECs: untreated (control), IL-1β and TNF-α-treated, MSC-EV-treated or IL-1β, TNF-α and MSC-EV-treated MCECs on Matrigel matrix. Scale bar represents 250 µm. (Right) Quantitative analysis of pro-angiogenic potential of MCECs presented as total segment length of capillaries per microscopic field at different time points (from 1 h until 8 h). D Viability of MCECs treated or untreated with MSC-EVs and cultured in microenvironment mimicking ischemic injury conditions by flow cytometry. MCECs were cultured in the presence of IL-1β and TNF-α under hypoxic conditions (2% O₂). Percent content of alive (AnnV⁻/PI⁻), necrotic (AnnV⁻/PI⁺), early apoptotic (AnnV⁺/PI⁻) and late apoptotic (AnnV⁺/PI⁺) cells at 4 h post MSC-EVs were shown. E Viability of MCECs stained with DAPI by flow cytometry. Student t-test, comparison with untreated control cells (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001)