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. 2022 Mar 25;12:5176. doi: 10.1038/s41598-022-08987-3

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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 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/.

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LYVE-1-expressing macrophages promote the migration and tube formation of human umbilical vein endothelial cells (HUVECs). A Lyve1-expression vector was transfected into RAW264.7 cells to generate Lyve1-expressing macrophages. (A) RT-PCR analysis of angiogenesis-related genes (Mmp2, Mmp9, and Vegfa) in RAW264.7 cells and Lyve1-expressing macrophages (mean ± SD, n = 3). (B) Quantification of VEGF-A in conditioned medium from Lyve1-expressing macrophages (CM-L) and RAW264.7 cells (CM-R) by ELISA (mean ± SD, n = 3). (C) Scratch wound healing test for HUVECs. The confluent cell monolayer of HUVECs was scratched with 200-μl pipette tips. Conditioned media (CM-L, CM-R), EBM-2 supplemented with EGM-2 (EBM/EGM), or serum-free EBM-2 (EBM) were added to culture plates. (D,E) Percentages of closure of scratched area at 24 h (mean ± SD, n = 4) and 36 h (median with interquartile range) after the initial scratch. Scratched areas were measured using an optimized plugin for ImageJ. (F) HUVEC tube formation assay. HUVECs were resuspended in conditioned media (CM-R, CM-L), EBM/EGM, or EBM. Cell suspension was seeded onto the solidified ECM gel and incubated for 6 h. Lower panels show the detection of tube-like structures of the corresponding upper panels using ImageJ. (G) The numbers of nodes and junctions and total tube length were quantified by ImageJ (mean ± SD, n = 4). Scale bars 300 µm (C) and 100 µm. Data were collected from 2–3 independent experiments to obtain consistent results. Data were statistically analyzed by unpaired Student’s t-test (Aa–c,B), one-way ANOVA and Tukey's post hoc test (D), Kruskal–Wallis test and Dunn's post hoc test (E), or Brown-Forsythe and Welch ANOVA test and Dunnett's T3 post hoc test (Ga–c). ∗p < 0.05, ∗ p < 0.01, and ∗∗∗p < 0.0001.