Noncaloric monosaccharides induce excessive sprouting angiogenesis in zebrafish via foxo1a-marcksl1a signal
- Affiliated Hospital of Nantong University, Nantong Laboratory of Development and Diseases, School of Life Science; Co-innovation Center of Neuroregeneration, Nantong University, China
- Suqian First Hospital, China
- Medical School, Nantong University, China
Figures
Figure 1 with 5 supplements
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 intersegmental vessels (ISVs) and dorsal lateral anastomotic vessels (DLAVs). (d) Statistical analysis of the total length of ISVs in control and glucose-treated embryos (n=8). 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 (n=8). 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 (n=8). 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 (n=8). 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.
Figure 1—figure supplement 1
Confocal imaging analysis of Tg(fli1aEP:EGFP-CAAX)ntu666 embryos at 48 hr post fertilization (hpf) and 72 hpf.
Figure 1—figure supplement 2
The diagrams show the glucose treatment time window and imaging time point.
Figure 1—figure supplement 3
Total glucose concentrations at different development stages in control and high glucose-treated embryos.
(a) A diagram showing the glucose treatment time window and concentration measuring time point. (b) Statistical analysis of the glucose concentration in control and high glucose-treated embryos. One-way ANOVA, ****p<0.0001.
Figure 1—figure supplement 4
Stereo microscopic analysis of control, glucose, and sucrase-treated embryos in a bright field.
(a–c) Imaging analysis of control, glucose, and sucrase-treated embryos in bright field. (d) A diagram showing the sucrase treatment time window and imaging time point. (e) A diagram indicating the imaging positions of the zebrafish embryos. (f) Confocal imaging analysis of the control and sucrase-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos. The red bar indicates position 1; the green bar indicates the position. (g) A diagram showing the sucrase treatment time window and imaging time point. (h) Confocal imaging analysis of the control and sucrase-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos. The red bar indicates position 1; the green bar indicates the position.
Figure 1—figure supplement 5
Confocal imaging analysis of 1–4% glucose-treated blood vessels.
(a) A diagram showing the glucose treatment time window and imaging time point. (b–g’) Confocal imaging analysis of the control, 1%, 2%, 3%, and 4% glucose-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos.
Figure 2 with 4 supplements
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 intersegmental vessels (ISVs). (h) Statistical analysis of the total length of ISVs in control and monosaccharides-treated embryos (n=5). (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.
Figure 2—figure supplement 1
Fructose treatment caused excessive angiogenesis in zebrafish.
(a) A diagram showing the fructose treatment time window and imaging time point. (b) A diagram indicating the imaging position of the zebrafish embryos. (c, d) Confocal imaging analysis of the control and glucose-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos. (e, f) Imaging analysis of control and fructose-treated embryos in bright field.
Figure 2—figure supplement 2
Lactose and maltose treatment did not cause excessive angiogenesis in zebrafish.
(a) A diagram showing the lactose and maltose treatment time window and imaging time point. (b–d’’) Confocal imaging analysis of the control, lactose-, and maltose-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos.
Figure 2—figure supplement 3
Higher concentration sucrase and maltose treatment cause excessive angiogenesis in zebrafish embryos.
(a) A diagram showing the sucrase and maltose treatment time window and imaging time point. (b) Confocal imaging analysis of the control and sucrase-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos. (c) Statistical analysis of the total length of intersegmental vessels (ISVs) in control and sucrase-treated embryos (n=5). t-test, ****p<0.0001. (d) Confocal imaging analysis of the control and maltose-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos.
Figure 2—figure supplement 4
Pyruvic acid treatment did not cause excessive angiogenesis in zebrafish.
(a) A diagram showing the pyruvic acid treatment time window and imaging time point. (b) Confocal imaging analysis of the pyruvic acid-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos. (c) A diagram showing the pyruvic acid treatment time window and imaging time point. (d) Confocal imaging analysis of the pyruvic acid-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos.
Figure 3
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)ntu666 embryos. 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 intersegmental vessel (ISV) in glucose-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos. Arrowheads indicate ectopic angiogenic sprouts. (h-j’) The feature plot of tip cell marker genes esm1, apln and cxcr4a of control and high glucose group in arterial and capillary ECs. (k) The violin plot of tip cell marker genes esm1, apln and cxcr4a of control and high glucose group in arterial and capillary ECs.
Figure 4 with 4 supplements
Single-cell transcriptome sequencing analysis of endothelial cells (ECs) 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 fluorescence-activated cell sorting (FACS) technology. (b) The measured cells were divided into six individual clusters based on gene expression profiles using UMAP. (c–h) The violin plots of some EC 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.
Figure 4—figure supplement 1
Overview of the number of genes, total Unique Molecular Identifiers (UMIs), and percentage of mitochondrial UMIs for the single-cell RNA sequencing.
(a) Before filtering. (b) After filtering. Cell selection criteria: 500<number of genes<3000; 0<percentage of mitochondrial UMIs<5%.
Figure 4—figure supplement 2
UMAP representation of endothelial cell (EC) subpopulations.
All single cells (after filtering) from the control and D-glucose treated were included in this illustration.
Figure 4—figure supplement 3
Transcriptome sequencing analysis of control, high D-glucose-, and high L-glucose-treated embryos.
(a) The heatmap of differentially expressed genes (DEGs). (b) Volcano map of DEGs after high D-glucose treatment. (c) Volcano map of DEGs after high L-glucose treatment.
Figure 4—figure supplement 4
The heatmap of metabolism-related genes.
(a) The heatmap of gluconeogenesis-related genes. (b) The heatmap of glycolysis-related genes. (c) The heatmap of oxidative phosphorylation-related genes.
Figure 5
Foxo1a was involved in the excessive angiogenesis induced by high glucose treatment.
(a) The volcano plot of differential expression genes in arterial and capillary endothelial cells (ECs). The avg_log2FC greater than 1 was considered significant, including 523 downregulated genes (blue dots) and 1201 upregulated genes (red dots). (b) Gene ontology (GO) analysis of 523 downregulated 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 intersegmental vessels (ISVs) in control embryos, AS1842856-treated embryos, and foxo1a MO-injected embryos (n=5). t-test, ****p<0.0001.
Figure 6
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)ntu666 embryos. (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 intersegmental vessels (ISVs) in control, high glucose-treated, hsp70l:foxo1a-P2A-mCherry, and high glucose-treated embryos (n=6), 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 and high glucose-treated embryos (n=9). t-test, ****p<0.0001. (j) Statistical analysis of the total length of ISVs in high glucose-treated embryos, and fli1EP:foxo1a-mApple and high glucose-treated embryos (n=8). t-test, ***p<0.001.
Figure 7 with 2 supplements
Marcksl1a overexpression induced excessive angiogenesis in zebrafish embryos.
(a) The violin plot of endothelial cells (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-time PCR analysis of marcksl1a expression in control, high glucose-, and high L-glucose-treated embryos (n=6). 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)ntu666 embryos. (g) Statistical analysis of the total length of intersegmental vessels (ISVs) in control and hsp70l:marcksl1a-P2A-mCherry-injected embryos (n=5). t-test, **p<0.01.
Figure 7—figure supplement 1
Multiple amino acid sequence alignment of mouse FOXO1 and zebrafish Foxo1a.
The DNA binding domain of mouse FOXO1 is demarcated with blue.
Figure 7—figure supplement 2
Confocal imaging analysis of blood vessels in the embryos with Lenvatinib treatment.
(a) A diagram showing the Lenvatinib treatment time window and imaging time point. (b) Confocal imaging analysis of the control and Lenvatinib-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos. (c) A diagram showing the Lenvatinib treatment time window and imaging time point. (d) Confocal imaging analysis of the control and Lenvatinib-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos.
Figure 8
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-time PCR analysis of marcksl1a expression in control and AS1842856-treated embryos (n=3). t-test, ***p<0.001. (c) Real-time PCR analysis of marcksl1a expression in control and foxo1a overexpressed embryos (n=3). 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 chromatin immunoprecipitation (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 (n=3), respectively. t-test, **p<0.01, ****p<0.0001. (h–m) Confocal imaging analysis of blood vessels in control, high glucose, high glucose and Lenvatinib, high glucose+marcksl1a MO, high L-glucose, and high L-glucose+marcksl1a MO groups. (n) Statistical analysis of the total length of intersegmental vessels (ISVs) in the groups in h–m (n=6), respectively. One-way ANOVA, ****p<0.0001.
Author response image 1
Videos
Video 1
The 3D structure of the blood vessels in the control Tg(fli1aEP:EGFP-CAAX)ntu666 embryos.
Video 2
The 3D structure of the blood vessels in the glucose-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos.
Video 3
Time-lapse imaging analysis of filopodia in the control Tg(fli1aEP:EGFP-CAAX)ntu666 embryos.
Video 4
Time-lapse imaging analysis of filopodia in the glucose-treated Tg(fli1aEP:EGFP-CAAX)ntu666 embryos.
Additional files
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Supplementary file 1
Cell proportions and numbers of each cluster.
- https://cdn.elifesciences.org/articles/95427/elife-95427-supp1-v1.xlsx
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Supplementary file 2
Cell proportions and numbers of each cluster.
- https://cdn.elifesciences.org/articles/95427/elife-95427-supp2-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/95427/elife-95427-mdarchecklist1-v1.docx
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Noncaloric monosaccharides induce excessive sprouting angiogenesis in zebrafish via foxo1a-marcksl1a signal
eLife 13:RP95427.
https://doi.org/10.7554/eLife.95427.3
