Effects of Ishige okamurae (IO) extract on zebrafish embryo. (A) Effects of IO extract on the survival rate of transgenic zebrafish (flk:EGFP) embryos. The embryos were treated with 10, 30, and 100 µg/mL of IO extract for 24, 48, 72, 96, 120, 144, and 168 h post fertilization (hpf). The treatment effects were normalized to blank (0 µg/mL IO extract). (B) The survival rate of transgenic zebrafish (flk:EGFP) embryos treated with 10 µg/mL IO extract and 130 mM glucose together. (C) Fluorescence microscopic images of the retinal vessels of transgenic zebrafish (flk:EGFP) embryos treated with IO extract. (D) The diameter of the hyaloid retinal vessel treated with IO extract. (E) Images of the whole body vessel formation in transgenic zebrafish (flk:EGFP) embryos treated with IO extract as obtained by fluorescence microscopy. (F) Quantified fluorescence intensity of the whole body treated with IO extract (a: 0 mM glucose + 0 µg/mL IO, b: 130 mM glucose + 0 µg/mL IO, and c: 130 mM glucose + 10 µg/mL IO). The effects of 130 mM glucose on vessel formation were compared with B (blank (0 mM glucose + 0 µg/mL IO extract)). The effects of Ishophloroglucin A (IPA) on high glucose-induced vessel formation were normalized to C (control (130 mM glucose + 0 µg/mL IO extract)). Scale bar (C) 20 µm, (E) 1000 µm. * p ˂ 0.05, ^#p ˂ 0.05.

Effects of IPA on transgenic zebrafish (flk:EGFP) embryos. (A) Effects of IPA on the survival rate of transgenic zebrafish (flk:EGFP) embryos. The embryos were treated with 0.3, 1.5, 3, and 5 µM IPA for 24, 48, 72, 96, 120, 144, and 168 hpf. The treatment effects were normalized to blank (0 µM IPA). (B) Fluorescence microscopic images of the retinal vessels of transgenic zebrafish (flk:EGFP) embryos treated with IPA. (C) The diameter of hyaloid retinal vessels treated with IPA. (D) The images of whole body vessel formation in transgenic zebrafish (flk:EGFP) embryos treated with IPA as obtained by fluorescence microscopy. (E) Quantified fluorescence intensity of the whole body treated with IO extract (a: 0 mM glucose + 0 µM IPA, b: 130 mM glucose + 0 µM IPA, c: 130 mM glucose + 0.05 µM IPA, d: 130 mM glucose + 0.15 µM IPA, e: 130 mM glucose + 0.5 µM IPA, and f: 130 mM glucose + 1.5 µM IPA). The effects of 130 mM glucose on vessel formation were compared to B (blank (0 mM glucose + 0 µM IPA)). The effects of IPA on high glucose-induced vessel formation were normalized to C (control (130 mM glucose + 0 µM IPA)). Scale bar (B) 20 µm, (D) 1000 µm. ns; not significant, * p ˂ 0.05, ** p ˂ 0.01, ^#p ˂ 0.05.

Effects of IPA on transgenic zebrafish (flk:EGFP) embryos. (A) Effects of IPA on the survival rate of transgenic zebrafish (flk:EGFP) embryos. The embryos were treated with 0.3, 1.5, 3, and 5 µM IPA for 24, 48, 72, 96, 120, 144, and 168 hpf. The treatment effects were normalized to blank (0 µM IPA). (B) Fluorescence microscopic images of the retinal vessels of transgenic zebrafish (flk:EGFP) embryos treated with IPA. (C) The diameter of hyaloid retinal vessels treated with IPA. (D) The images of whole body vessel formation in transgenic zebrafish (flk:EGFP) embryos treated with IPA as obtained by fluorescence microscopy. (E) Quantified fluorescence intensity of the whole body treated with IO extract (a: 0 mM glucose + 0 µM IPA, b: 130 mM glucose + 0 µM IPA, c: 130 mM glucose + 0.05 µM IPA, d: 130 mM glucose + 0.15 µM IPA, e: 130 mM glucose + 0.5 µM IPA, and f: 130 mM glucose + 1.5 µM IPA). The effects of 130 mM glucose on vessel formation were compared to B (blank (0 mM glucose + 0 µM IPA)). The effects of IPA on high glucose-induced vessel formation were normalized to C (control (130 mM glucose + 0 µM IPA)). Scale bar (B) 20 µm, (D) 1000 µm. ns; not significant, * p ˂ 0.05, ** p ˂ 0.01, ^#p ˂ 0.05.

IPA inhibits the proliferation of EA.hy926 cells. (A) Cytotoxicity of IPA in EA.hy926 cells. The cells were treated with different IPA concentrations (0, 0.05, 0.15, 0.5, 1.5, and 2.5 µM) for 24 h, and the cell viability was determined by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The results were normalized to blank (0 µM IPA). (B) IPA inhibits the proliferation of high glucose-induced EA.hy926 cells. The cells were treated with different concentrations of IPA (0.05, 0.15, 0.5, and 1.5 µM), along with 30 mM glucose. MTT assay was performed to determine cell viability. The anti-proliferative effects of IPA in high glucose-induced cells were normalized to C (control (30 mM glucose + 0 µM IPA)), and the effects of 30 mM glucose were compared to B (blank (0 mM glucose + 0 µM IPA)). ns; not significant, * p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001, ^##p ˂ 0.01.

(A) IPA inhibits high glucose-induced cell migration. The cells were treated with different concentrations of IPA (0.15, 0.5, and 1.5 µM), along with 30 mM glucose. The cell monolayer was scraped at the middle of the well, and the initial gap length (0 h) and the final gap length (12 h) was measured. (B) The gap closure was quantified by percentage (%). (C) IPA inhibits cell migration through transwell filter chambers. The migrated cells were fixed, stained, and counted. (D) Quantification of migrated cells through transwell filter chambers. Matrix Metalloproteinase (MMP) (MMP-2 and -9) expression levels were evaluated using commercial Enzyme-Linked Immunosorbent Assay (ELISA) kits. (E) Quantification of MMP-2 expression. (F) Quantification of MMP-9 expression. The effects of 30 mM glucose on cell migration/MMP expression levels were compared to B (blank (0 mM glucose + 0 µM IPA)), and the effects of IPA on high glucose-induced cell migration/MMP expression level were normalized to C (control (30 mM glucose + 0 µM IPA)). Scale bar (A) and (C) 1000 µm. ns; not significant, * p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001, ^##p ˂ 0.01, ^###p ˂ 0.001.

(A) IPA inhibits high glucose-induced cell migration. The cells were treated with different concentrations of IPA (0.15, 0.5, and 1.5 µM), along with 30 mM glucose. The cell monolayer was scraped at the middle of the well, and the initial gap length (0 h) and the final gap length (12 h) was measured. (B) The gap closure was quantified by percentage (%). (C) IPA inhibits cell migration through transwell filter chambers. The migrated cells were fixed, stained, and counted. (D) Quantification of migrated cells through transwell filter chambers. Matrix Metalloproteinase (MMP) (MMP-2 and -9) expression levels were evaluated using commercial Enzyme-Linked Immunosorbent Assay (ELISA) kits. (E) Quantification of MMP-2 expression. (F) Quantification of MMP-9 expression. The effects of 30 mM glucose on cell migration/MMP expression levels were compared to B (blank (0 mM glucose + 0 µM IPA)), and the effects of IPA on high glucose-induced cell migration/MMP expression level were normalized to C (control (30 mM glucose + 0 µM IPA)). Scale bar (A) and (C) 1000 µm. ns; not significant, * p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001, ^##p ˂ 0.01, ^###p ˂ 0.001.

(A) IPA suppresses high glucose-induced capillary formation in Matrigel^®. The cells were seeded on Matrigel^® with different concentrations of IPA (0.15, 0.5, and 1.5 µM), along with 30 mM glucose including B (blank (0 mM glucose + 0 µM IPA)) and C (control (30 mM glucose + 0 µM IPA)). After 6 h incubation, the cells were photographed and the angiogenic score was determined. (B) Quantification of capillary formation. The effects of 30 mM glucose on capillary formation were compared to B (blank (0 mM glucose + 0 µM IPA)). The effects of IPA on high glucose-induced capillary formation were normalized to C (control (30 mM glucose + 0 µM IPA)). Scale bar (A) 1000 µm. * p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001, ^###p ˂ 0.001.

(A) IPA suppresses high glucose-induced capillary formation in Matrigel^®. The cells were seeded on Matrigel^® with different concentrations of IPA (0.15, 0.5, and 1.5 µM), along with 30 mM glucose including B (blank (0 mM glucose + 0 µM IPA)) and C (control (30 mM glucose + 0 µM IPA)). After 6 h incubation, the cells were photographed and the angiogenic score was determined. (B) Quantification of capillary formation. The effects of 30 mM glucose on capillary formation were compared to B (blank (0 mM glucose + 0 µM IPA)). The effects of IPA on high glucose-induced capillary formation were normalized to C (control (30 mM glucose + 0 µM IPA)). Scale bar (A) 1000 µm. * p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001, ^###p ˂ 0.001.

(A) IPA inhibits vascular endothelial growth factor receptor 2 (VEGFR-2) and its downstream signaling molecules. IPA attenuating high glucose-induced phosphorylation of VEGFR-2 and downstream signaling molecules extracellular signal-regulated kinase (ERK), protein kinase B (AKT), c-Jun N-terminal kinase (JNK), and endothelial nitric oxide synthase (eNOS) were detected using western blotting. (B) Quantitative evaluation of the protein expression. The effects of 30 mM glucose on the expression of each protein were compared to B (blank (0 mM glucose + 0 µM IPA)). The effects of IPA on high glucose-induced protein expression were normalized to C (control (30 mM glucose + 0 µM IPA)). ns; not significant, * p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001, ^#p ˂ 0.05, ^##p ˂ 0.01, ^###p ˂ 0.001.

(A) IPA inhibits vascular endothelial growth factor receptor 2 (VEGFR-2) and its downstream signaling molecules. IPA attenuating high glucose-induced phosphorylation of VEGFR-2 and downstream signaling molecules extracellular signal-regulated kinase (ERK), protein kinase B (AKT), c-Jun N-terminal kinase (JNK), and endothelial nitric oxide synthase (eNOS) were detected using western blotting. (B) Quantitative evaluation of the protein expression. The effects of 30 mM glucose on the expression of each protein were compared to B (blank (0 mM glucose + 0 µM IPA)). The effects of IPA on high glucose-induced protein expression were normalized to C (control (30 mM glucose + 0 µM IPA)). ns; not significant, * p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001, ^#p ˂ 0.05, ^##p ˂ 0.01, ^###p ˂ 0.001.