Epidemiology and Global Health

Noncaloric monosaccharides induce excessive sprouting angiogenesis in zebrafish via foxo1a-marcksl1a signal

Xiaoning Wang
Jinxiang Zhao
Jiehuan Xu
Bowen Li
Xia Liu author has email address
Gangcai Xie author has email address
Xuchu Duan author has email address
Dong Liu author has email address

Affiliated Hospital of Nantong University, Nantong Laboratory of Development and Diseases, School of Life Science; Co-innovation Center of Neuroregeneration, Nantong University, Nantong, China
Suqian First Hospital, Suqian, China
Medical School, Nantong University, Nantong, China

https://doi.org/10.7554/eLife.95427.2

Open access
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Figures and data

Glucose treatment caused excessive angiogenesis in zebrafish.
a, A diagram showing the glucose treatment time window and imaging time point. b, A diagram indicating the imaging positions of the zebrafish embryos. c, Confocal imaging analysis of the control and glucose-treated embryos. The red bar indicates position 1; the green bar indicates position 2. Arrowheads indicate the ectopic branching from the dorsal aorta. Stars indicate the ectopic vessels from ISVs and DLAVs. d, Statistical analysis of the total length of ISVs in control and glucose-treated embryos. t-test, ****p<0.0001. e, A diagram showing the glucose treatment time window and imaging time point. f, Confocal imaging analysis of the control and glucose-treated embryos. The red bar indicates position 1; the green bar indicates position 2. Arrowheads indicate the ectopic branching from the dorsal aorta. Stars indicate the ectopic vessels from ISVs and DLAVs. g, Statistical analysis of the total length of ISVs in control and glucose-treated embryos. t-test, ****p<0.0001. h, A diagram showing the glucose treatment time window and imaging time point. i, Confocal imaging analysis of the control and glucose-treated embryos. The red bar indicates position 1; the green bar indicates position 2. Arrowheads indicate the ectopic branching from the dorsal aorta. Stars indicate the ectopic vessels from ISVs and DLAVs. j, Statistical analysis of the total length of ISVs in control and glucose-treated embryos. t-test, ****p<0.0001. k, A diagram showing the glucose treatment time window and imaging time point. l, Confocal imaging analysis of the control and glucose-treated embryos. The red bar indicates position 1; the green bar indicates position 2. Arrowheads indicate the ectopic branching from the dorsal aorta. Stars indicate the ectopic vessels from ISVs and DLAVs. m, Statistical analysis of the total length of ISVs in control and glucose-treated embryos. t-test, ****p<0.0001. o, A diagram showing the blood vessels in position 2 indicated in panel b of control embryos. p, A diagram showing the blood vessels in position 2 indicated in panel b of high glucose-treated embryos.

L-glucose and mannose treatment caused excessive angiogenesis as well.
a, A diagram showing the monosaccharides treatment time window and imaging time point. b, A diagram indicating the imaging position of the zebrafish embryos. c-g, Confocal imaging analysis of the control and monosaccharides, including L-glucose, D-mannose, D-ribose, and L-arabinose, treated embryos. Arrowheads indicate the ectopic branching from the dorsal aorta. Stars indicate the ectopic vessels from ISVs. h, Statistical analysis of the total length of ISVs in control and monosaccharides treated embryos. i-j, Confocal imaging analysis of the control and glucose-treated embryos. Arrowheads indicate the ectopic branching of arteries. Stars indicate the ectopic branching of veins. t-test, ****p<0.0001.

High glucose treatment induced endothelial differentiation into tip cell-like cells.
a, A diagram showing the confocal time-lapse imaging time window. b, A diagram indicating the imaging position of the zebrafish embryos. c, Confocal time-lapse imaging analysis of blood vessels in control Tg(fli1aEP:EGFP-CAAX)^ntu666 embryos. d, A diagram showing the glucose treatment time window and confocal time-lapse imaging time window. e, Confocal time-lapse imaging analysis of blood vessels in glucose-treated Tg(fli1aEP:EGFP-CAAX)^ntu666 embryos. Arrowheads indicate the ectopic angiogenic branches. f, A snapshot of confocal time-lapse imaging analysis of blood vessels in glucose-treated Tg(fli1aEP:EGFP-CAAX)^ntu666embryos. Z stacks were used to make 3D color projections, where blue represents the most proximal (closest to the viewer), and red represents the most distal (farthest from the viewer). Arrowheads indicate ectopic angiogenic sprouts. g, A snapshot of confocal time-lapse imaging analysis of an ISV in glucose-treated Tg(fli1aEP:EGFP-CAAX)^ntu666 embryos. Arrowheads indicate ectopic angiogenic sprouts.

Single-cell transcriptome sequencing analysis of endothelial cells in control and high glucose treated embryos.
a, Schematic diagram of the single-cell sequencing process. 300 embryos in the control group and 300 in the high glucose group were used, and ECs were sorted by GFP fluorescent using FACS technology. b, The measured cells were divided into 6 individual clusters based on gene expression profiles using UMAP. c-h, The violin plots of some endothelial cell marker genes. i, The proportion of ECs in each cluster of control and high glucose groups. j, Changes of ECs percentage in arterial and capillary ECs, endocardium, and proliferating ECs of control and high glucose group.

Foxo1a was involved in the excessive angiogenesis induced by high glucose treatment.
a, The volcano plot of differential expression genes in arterial and capillary ECs. The avg_log2FC greater than 1 was considered significant, including 523 down-regulated genes (blue dots) and 1201 up-regulated genes (red dots). b, GO analysis of 523 down-regulated genes in arterial and capillary ECs. c, The feature plot of ECs marker gene pecam1 of control and high glucose group in arterial and capillary ECs. c’, The violin plot of ECs marker gene pecam1 of control and high glucose group in arterial and capillary ECs. d, The feature plot of gene foxo1a of control and high glucose group in arterial and capillary ECs. d’, The violin plot of gene foxo1a of control and high glucose group in arterial and capillary ECs. e, Average expression of gene pecam1 and foxo1a in control and high glucose group. f, Whole-mount in situ hybridization analysis of foxo1a in control, high glucose, and high L-glucose treated embryos. g, A diagram showing the foxo1 inhibitor treatment time window. h, Confocal imaging analysis of control embryos, AS1842856 treated embryos and foxo1a MO-injected embryos. Arrowheads indicate ectopic angiogenic sprouts. i, Statistical analysis of the total length of ISVs in control embryos, AS1842856 treated embryos, and foxo1a MO-injected embryos. t-test, ****p<0.0001.

Foxo1a gain of function can partially rescue the excessive angiogenesis induced by high glucose treatment.
a, A diagram showing the high glucose treatment time window and heat shock treated time point. b, Confocal imaging analysis of blood vessels in control Tg(fli1aEP:EGFP-CAAX)^ntu666embryos. c, Confocal imaging analysis of blood vessels in high glucose treated Tg(fli1aEP:EGFP-CAAX)^ntu666 embryos. d-d’, Confocal imaging analysis of blood vessels in hsp70l:foxo1a-P2A-mCherry and high glucose treated Tg(fli1aEP:EGFP-CAAX)^ntu666 embryos. e, Statistical analysis of the total length of ISVs in control, high glucose-treated, hsp70l:foxo1a-P2A-mCherry and high glucose treated embryos, respectively. one-way ANOVA, ****p<0.0001. f, A diagram showing the high glucose treatment time window and imaging time point. g, Confocal imaging analysis of blood vessels in high glucose treated Tg(fli1aEP:EGFP-CAAX)^ntu666 embryos. h, Confocal imaging analysis of blood vessels in fli1EP:foxo1a-mApple and high glucose treated Tg(fli1aEP:EGFP-CAAX)^ntu666 embryos. i, Statistical analysis of the number of sprouts per ISV in high glucose treated embryos, and fli1EP:foxo1a-mApple & high glucose treated embryos. t-test, ****p<0.0001. j, Statistical analysis of the total length of ISVs in high glucose treated embryos, and fli1EP:foxo1a-mApple & high glucose treated embryos. t-test, ***p<0.001.

Marcksl1a over-expression induced excessive angiogenesis in zebrafish embryos.
a, The violin plot of ECs marker gene kdrl of control and high glucose group in arterial and capillary ECs. b, The violin plot of gene marcksl1a of control and high glucose group in arterial and capillary ECs. c, Real-rime PCR analysis of marcksl1a expression in control, high glucose, and high L-glucose treated embryos. t-test, ****p<0.0001. d, Whole-mount in situ hybridization analysis of marcksl1a in control, high glucose, and high L-glucose treated embryos. e-f’, Confocal imaging analysis of blood vessels in control and hsp70l:marcksl1a-P2A-mCherry injected Tg(fli1aEP:EGFP-CAAX)^ntu666embryos. g, Statistical analysis of the total length of ISVs in control and hsp70l:marcksl1a-P2A-mCherry injected embryos. t-test, **p<0.01.

Noncaloric monosaccharides induced excessive angiogenesis through foxo1a-marcksl1a signal in zebrafish embryos.
a, A diagram showing the Foxo1 inhibitor and heat shock treatment time window. b, Real-rime PCR analysis of marcksl1a expression in control and AS1842856 treated embryos. t-test, ***p<0.001. c, Real-rime PCR analysis of marcksl1a expression in control and foxo1a overexpressed embryos. t-test, **p<0.01. d, A sequence logo of Foxo1 binding sequence presented in the JASPAR database (https://jaspar.genereg.net/) and two candidate binding sites at the upstream of transcription start site (TSS) of marcksl1a in zebrafish. e, Results of the ChIP-PCR assay indicated that BS1 and BS2 are Foxo1a-binding sites of marcksl1a in zebrafish. Input sonicated genomic DNA samples without immunoprecipitation as a positive control. IgG, sonicated, and IgG-immunoprecipitated genomic DNA samples as a negative control. f-g, Luciferase reporter activity in foxo1a overexpressed or knocked down embryos, respectively. t-test, **p<0.01, ****p<0.0001. h-m, Confocal imaging analysis of blood vessels in control, high glucose, high glucose & Lenvatinib, high glucose+marcksl1a MO, high L-glucose and high L-glucose+marcksl1a MO groups. n, Statistical analysis of the total length of ISVs in the groups in figure h-m, respectively. one-way ANOVA, ****p<0.0001.

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