In silico prediction and biological assessment of novel angiogenesis modulators from traditional Chinese medicine

Figures

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  FIGURE 1. This study compromises three main parts. (A) Literature assessment of angiogenesis-modulating individual metabolites from TCM, (B) Decision tree and clustering analysis to validate known mechanisms, and (C) Identification and assessment of new small-molecule modulators of angiogenesis in vitro and in vivo.

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  FIGURE 2. Protein bioactivities predicted by PIDGIN between individual TCM metabolites and most enriched targets. Individual metabolites were clustered based on the profile of target prediction (C1–C4); pink and green represent literature evidence for inhibition (INHIBIT) and promotion (PROMOTE) of angiogenesis, respectively, whilst the enriched anti-angiogenic (INHIBIT) and pro-angiogenic (PROMOTE) protein targets are shown in the MoA column on the right, respectively.

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  FIGURE 3. Pathway enrichment signatures for individual pro-angiogenic (Promote) or anti-angiogenic (Inhibit) metabolites derived from TCM. Individual metabolites was clustered into five groups labelled with different colours (Cluster 1–5).

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  FIGURE 4. Mode of action analysis of individual pro- and anti-angiogenic metabolites derived from TCM, based on the most important ten predicted targets and DEGs. The number of metabolites in each node is shown, along with the entropy (the information gain) to denote the quality of each split in the tree. Colour is used to indicate the purity for PROMOTER (blue) or INHIBITOR (orange) classification of metabolites. It can be seen that both DEGs and Target prediction were selected by the decision tree as important features that can distinguish between inhibitors and promoters.

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  FIGURE 5. Modulation of endothelial tube formation by ferulic acid (FA) and curculigostide (CUR) in vitro. In a HUVEC-HDF co-culture model, ferulic acid stimulated endothelial tube formation at 10 nM but produced an inhibitory effect at higher concentrations. Curculigostide stimulated endothelial tube formation at 10 nM–1 μM. VEGF was included as the positive control. Statistical analyses were performed by one‐way ANOVA followed by Dunnett’s post hoc test. Data represents mean ± SEM (n = 4–6). Compared with the control group, *p < 0.05, **p < 0.01, ***p < 0.001.

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  FIGURE 6. Ferulic acid (FA) and curculigostide (CUR) stimulated angiogenesis in a zebrafish model. Lateral view of zebrafish embryos treated with DMSO (0.1%, Control), PTK787 (0.2 μg/mL), PTK 787 plus FA (10, 20, 40, 80, and 160 μM) or CUR (10, 20, 40, 80, and 160 μM) for 24 h. FA (at 40, 80, and 160 μM) and CUR (at 160 μM) restored angiogenesis in ISVs against PTK787‐induced impairment. Statistical analysis data were shown as mean ± SEM (n ≥ 8). The comparison between groups was performed by student’s test. ## p < 0.01 versus Control group; **p < 0.01 versus PTK787‐treated group.

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  FIGURE 7. Lack of anti-angiogenic effects of deoxycholic acid (DA) and ursodeoxycholic acid (UA) in a zebrafish model. Lateral view of zebrafish embryos treated with DMSO (0.1%, Control), PTK787 (0.2 μg/mL), two bile acids added for 24 h. At 16 μM of each bile acids, some deformity was apparent, with the degree of malformation (yolk sac edema, tail malformation) increased at 20 μM. Compared with the Control group, these two bile acids had no effects on angiogenesis in the ISVs. In contrast, PTK787 effectively inhibited the growth of the ISVs. Statistical analysis data were shown as mean ± SEM (n ≥ 8). The comparison between groups was performed by student’s test. ## p < 0.01 versus Control group.

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  FIGURE 8. Modulation of endothelial tube formation in vitro by tetrahydropalmatine (TET), 1 beta-hydroxyalantolactone (BHA), cinobufotalin (CIN) and isoalantolactone (ISO) selected by the Machine Learning model. Total tube area was shown. Statistical analyses were performed by one‐way ANOVA followed by Dunnett’s post hoc test. Data represents mean ± SEM (n = 4–6). Compared with the control group, * p < 0.05, ** p < 0.01, *** p < 0.001.

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  FIGURE 9. 1-beta-hydroxyalantolactone (BHA) and Cinobufotalin (CIN) stimulated angiogenesis in a zebrafish model. Lateral view of zebrafish embryos treated with DMSO (0.1%, Control), PTK787 (0.2 μg/mL), PTK 787 plus ginsenoside Rg1 (Rg1 40 μM, positive control), or PTK 787 plus the test individual metabolites from TCM (5, 10, 20, 40, and 80 μM) for 24 h. 1-beta-hydroxyalantolactone (BHA) and cinobufotalin (CIN) dose-dependently restored angiogenesis in ISVs against PTK787‐induced impairment. Tetrahydeopalmatine (TET) worsened the effect of PTK787 and isoalantolactone (ISO) caused lethality at 10 μM and higher concentrations. Quantification of total length of ISVs in different groups from three independent experiments. Statistical analysis data were shown as mean ± SEM (n ≥ 8). The comparison between groups was performed by student’s test. ## p < 0.01 versus Control group; **p < 0.01 versus PTK787‐treated group.

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  FIGURE 10. Ginsenosides Rb3 and Rc stimulated angiogenesis in a zebrafish model. Lateral view of zebrafish embryos treated with DMSO (0.1%, Control), PTK787 (0.2 μg/mL), PTK 787 plus ginsenoside Rb3 (1, 5, 10, 50, and 100 μg/mL) or Rc (1, 10, 50, 80, and 100 μg/mL) for 24 h Rb3 (100 μg/mL) and Rc (80 and 100 μg/mL) restored angiogenesis in ISVs against PTK787‐induced impairment. Quantification of total length of ISVs in different groups from three independent experiments. Statistical analysis data were shown as mean ± SEM (n ≥ 4). The comparison between groups was performed by student’s test. ## p < 0.01 versus Control group; **p < 0.01 versus PTK787‐treated group.