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. 2020 Jul 7;10:11186. doi: 10.1038/s41598-020-68019-w

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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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Inhibition of EP1, EP2 and EP4 restores pericyte–EC interaction. (a) RT-qPCR analysis of EP receptor expression profile in HBVPs. The different primer sets were validated for an efficiency of 2 ± 5%. EP3 expression was undetectable. (b) The effect of PGE2 on EP1, EP2 and EP4 expression in HBVPs was assessed by RT-qPCR 72 h post-treatment. The mRNA expression level of each EP was normalized to that of cyclophilin A using the delta-delta Ct method. Fold change relative to the control is shown. (c) HBVPs were treated with a cocktail of EP1 (ONO-8713), EP2 (PF-04418948) and EP4 (ONO-AE3-208) inhibitors (1 μM each) in DMSO or DMSO vehicle alone, then 5 h later with 100 nM PGE2 in DMSO or DMSO alone. Cell morphology was observed 72 h later. Scale bar 50 μm. (d) HBVPs (green) subjected to the same treatments as described in c were co-cultured on Matrigel with HUVECs (red) for 18 h. Scale bar 50 μm. The branching index (number of junctions/mm²), average vessel length and number of vessels were measured using Angiotool and ImageJ. One-way ANOVA test, *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.001; ns, not significant.