Figure 4.

Foxo1 (forkhead box protein O1) deficiency promoted human retinal microvascular endothelial cell (HRMEC) migration and resulted in the aggregation of endothelial cell (EC) nuclei in zebrafish embryos. A, Schematic diagram of drug treatment time window and confocal imaging region. B, Confocal imaging analysis of control (Ctrl), high glucose–treated, and AS1842856-treated Tg(fli1a:nGFP::kdrl:ras-mCherry) embryos at 72 hours post-fertilization (hpf). C, Statistics of the EC nuclei nearest neighbor distance (NND) in Ctrl (n=22), high glucose–treated (n=32), and AS1842856-treated embryos (n=26). t test. D, Confocal imaging analysis of EC nuclei in ISVs in the Ctrl embryos, high glucose–treated embryos, and Tg(hsp70l:foxo1a-6×His-P2A-mCherry) and high glucose–treated embryos at 72 hpf. E, Statistical analysis of the EC nuclei NND in the Ctrl embryos (n=22), high glucose–treated embryos (n=32), and Tg(hsp70l:foxo1a-6×His-P2A-mCherry) and high glucose–treated embryos (n=26) at 72 hpf. One-way ANOVA. F through H′, The results of the wound-healing assay showed that high glucose and AS1842856 treatment promotes HRMEC migration, in comparison with the dimethyl sulfoxide (DMSO)–treated group. I, Real-time PCR analysis of FOXO1 expression in HRMECs treated with DMSO and high glucose. Each dot represents data from an independent experiment (n=3). t test. J, Statistical analysis of the wound-healing percentage in the DMSO, high glucose–treated, and AS1842856-treated groups. Each dot represents data from an independent experiment (n=6). t test. Scale bars, 100 µm. nGFP indicates green fluorescent protein for nuclei.