Ibrutinib had potent toxicity in zebrafish embryos. (A) Ibrutinib induced a developmental deficiency in treated embryos at 72 h post-fertilization (hpf); a shorter body length, pericardial edema, and arrested blood flow were observed. Scale bar: 300 μm. (B) Heart rate of ibrutinib-treated embryos at 72 hpf; n = 10 for each group. (C) Survival curves of embryos treated with DMSO or ibrutinib at 12 hpf, survival rate was calculated from three independent experiments; n = 60 for each group in total. Asterisks indicate significant differences (DMSO vs. ibrutinib), **p < 0.01, ***p < 0.001.

Vascular formation was perturbed in ibrutinib-treated embryos. (A) Confocal images of blood vessels in the brain and trunk of ibrutinib-treated Tg (kdrl:EGFP) embryos at 72 h post-fertilization (hpf). Scale bar: 100 μm. (B) The percentage of normal intersegmental vessels (ISVs) in embryos presented at (A); n = 10 for each group. (C) Confocal images of blood vessels in the trunk of embryos treated and washed at different stages. Scale bar: 100 μm. (D) The percentage of normal ISVs in embryos presented at (C); n = 10 for each group. Asterisks indicate significant differences (DMSO vs. ibrutinib), ***p < 0.001.

Vascular lumens were collapsed after ibrutinib treatment. (A) Confocal images of the dorsal aorta (DA) and posterior cardinal vein (PCV) in the trunk of ibrutinib-treated Tg (kdrl:EGFP) embryos at 72 h post-fertilization (hpf). Scale bar: 50 μm. (B) H&E staining of cross-sections containing the DA and PCV of treated embryos at 72 hpf. Scale bar: 20 μm. (C) Immunofluorescence staining of cross-sections containing the DA and PCV of treated embryos at 72 hpf. Scale bar: 10 μm. (D) Diameters of the DA and PCV in treated embryos at 72 hpf; n = 10 for each group. Asterisks indicate significant differences (DMSO vs. ibrutinib), ***p < 0.001.

Reduced proliferation and enhanced apoptosis of vascular endothelial cells (VECs) in ibrutinib-treated embryos. (A) An EdU assay showed proliferation signals in the VECs of ibrutinib-treated embryos at 36 h post-fertilization (hpf). (B) The number of proliferated VECs was counted in the dorsal aorta (DA), posterior cardinal vein (PCV), and caudal vein (CV), as well as ISVs; n = 10 for each group. (C) A TUNEL assay showed apoptosis signals in the VECs of ibrutinib-treated embryos at 60 hpf. (D) The number of apoptotic VECs was counted in the DA, PCV, and CV, as well as intersegmental vessels (ISVs); n = 10 for each group. Asterisks indicate significant differences (DMSO vs. ibrutinib), **p < 0.01, ***p < 0.001.

Ibrutinib exposure altered the expression of vascular development-related genes. (A) The expression pattern of vascular marker genes, fli1, flt1, and kdrl, was examined in ibrutinib-treated embryos by whole-mount in situ hybridization (WISH) at 24 h post-fertilization (hpf). Numbers at bottom left indicate the number of embryos with similar staining pattern among all embryos examined (Fisher’s exact tests, p < 0.001). (B) The qRT-PCR results showed the expression of VEGFR genes, flt1, flt4, kdr, and kdrl, in ibrutinib-treated embryos at 24 and 48 hpf. Asterisks indicate significant differences (DMSO vs. ibrutinib), **p < 0.01, ***p < 0.001.