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. 2022 May 12;13:2594. doi: 10.1038/s41467-022-30197-8

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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. 3 — a Confocal z-projection images of on-chip angiogenic branches loaded without (left) or with IP (right) for 3 days. Green, VE-cadherin; blue, DAPI. b, d Confocal z-projection images of upstream (Up) and downstream (Down) injured vessels [b skin vessels in adult Tg(kdrl:EGFP) zebrafish; d aISVs in 3 dpf Tg(fli1a:EGFP) larva]. c, e Outer diameters (O.D.) of the upstream (red) and downstream (blue) injured vessels as in b, d are shown as percentages relative to that of pre-injured vessels. The x-axis shows the distance from the leading edge (arrows in b, d). Thin projections (arrowheads in b, d) are excluded from measurement. Data are means ± s.e.m. [c, upstream and downstream, n = 23 and 19 vessels examined over 10 and 11 animals, except for upstream at 50 μm (n = 22) and downstream at 30 μm (n = 18); d, upstream and downstream, n = 6 animals for each, except for upstream at 40 μm (n = 4) and downstream at 40 μm (n = 5)]. f Effects of changes in blood flow on the morphology of injured aISVs. Images of pre- (Pre-injury) and post-injured (Post-injury) aISVs in 3 dpf Tg(fli1a:Myr-EGFP) larva and its subsequent images at 3 hpi (Flow), after tricaine-induced blood flow arrest (Flow arrest), and after restarting blood flow (Flow restart). g Outer diameters of injured aISVs at 10 µm from the leading edge are as in c [upstream and downstream at Flow (n = 9 and 10 animals), Flow arrest (n = 9 and 10 animals), Flow restart (n = 8 and 9 animals)]. h DIC images of on-chip angiogenic branches before and 1 day after loading without (Control) and with IP (Intra.), EP (Extra.), or both types of pressure (Intra./Extra.). i Quantification of branch lengths as in h. Normalized values relative to that before pressure loading are means ± s.e.m. (the number of branches examined over 4 independent experiments: Control, n = 68 and 67; IP, n = 40 and 38; EP, n = 50 and 50; IP/EP, n = 50 and 50, for Days 0 and 1). j DIC images of IP-loaded angiogenic branch (Pre) and, subsequently, an unloaded vessel (Post) and its subsequent time-lapse images at elapsed times indicated at top. k Confocal z-projection images merged with bright-field images of an angiogenic branch, with a small population of RFP-expressing ECs, before (Pre) and after (Post) IP loading. Arrows, elongation direction; red, RFP; blue, Hoechst 33342. Right, binary RFP images. l–n Changes in Short- and Long-axis lengths of cells (m) and those in Short-axis length and Cell thickness of cells (n) upon IP loading were measured as in l, and expressed as a ratio of the values before and after IP loading. Each dot represents an individual cell. Right, binary images of RFP-expressing cells. Boxed areas are enlarged on the right (a, f) and top (j). Statistical significance was determined by two-way ANOVA followed by Sidak’s test (c, e), Kruskal–Wallis followed by Dunn’s test among the changes in the upstream or downstream vessel (g), and two-sided t test (i). *p < 0.05, **p < 0.01. Source data are provided as a Source data file. Scale bars: 100 µm (a, h), 10 µm (b, d, f), 50 µm (j), 25 µm (k, m, n).