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Decursin inhibits retinal neovascularization via suppression of VEGFR-2 activation.

Kim JH, Kim JH, Lee YM, Ahn EM, Kim KW, Yu YS - Mol. Vis. (2009)

Bottom Line: We evaluated the inhibitory effect of decursin on retinal neovascularization.Decursin significantly inhibited VEGF-induced proliferation, migration, and the formation of capillary-like networks of retinal endothelial cells in a dose-dependent manner.Moreover, the upregulation of glial fibrillary acidic protein expression was not detected in Mueller cells.

View Article: PubMed Central - PubMed

Affiliation: Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Department of Ophthalmology, College of Medicine, Seoul National University and Seoul Artificial Eye Center Clinical Research Institute, Seoul National University Hospital, Seoul 151-744, Korea.

ABSTRACT

Purpose: Pathologic angiogenesis in the retina leads to the catastrophic loss of vision. Retinopathy of prematurity (ROP), a vasoproliferative retinopathy, is a leading cause of blindness in children. We evaluated the inhibitory effect of decursin on retinal neovascularization.

Methods: Anti-angiogenic activity of decursin was evaluated by vascular endothelial growth factor (VEGF)-induced proliferation, migration, and in vitro tube formation assay of human retinal microvascular endothelial cells (HRMECs). We also used western blot analysis to assess inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) phosphorylation by decursin. After intravitreal injection of decursin in a mouse model of ROP, retinal neovascularization was examined by fluorescence angiography and vessel counting in cross-sections. The toxicity of decursin was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HRMECs as well as histologic and immunohistochemistry examination for glial fibrillary acidic protein in the retina.

Results: Decursin significantly inhibited VEGF-induced proliferation, migration, and the formation of capillary-like networks of retinal endothelial cells in a dose-dependent manner. Decursin inhibited VEGF-induced phosphorylation of VEGFR-2, blocking the VEGFR-2 signaling pathway. When intravitreously injected, decursin dramatically suppressed retinal neovascularization in a mouse model of ROP. Even in a high concentration, decursin never induced any structural or inflammatory changes to cells in retinal or vitreous layers. Moreover, the upregulation of glial fibrillary acidic protein expression was not detected in Mueller cells.

Conclusions: Our data suggest that decursin may be a potent anti-angiogenic agent targeting the VEGFR-2 signaling pathway, which significantly inhibits retinal neovascularization without retinal toxicity and may be applicable in various other vasoproliferative retinopathies as well.

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Decursin inhibits VEGF-induced migration and tube formation of HRMECs. A: Human retinal microvascular endothelial cells (HRMECs) were treated with 20 ng/ml vascular endothelial growth factor (VEGF) or 1–50 µM decursin for 24 h. A cell-proliferation assay with [3H]-thymidine was performed. Each value represents the mean (±SD) of three independent experiments. The asterisk indicates a p<0.05. B: HRMECs were treated with 20 ng/ml VEGF or 10 µM decursin for 5 min. Western blot analysis using phospho- (p-)VEGFR-2 and vascular endothelial growth factor receptor-2 (VEGFR-2) antibodies was performed, with β-actin serving as the loading control. Figures were selected as representative data from three independent experiments. Quantitative analysis was performed by measuring the intensity relative to the control. Each value represents means±SEM from three independent experiments. The asterisk indicates a p<0.05. The size of scale bars in figure A, B were 100 µm.
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f1: Decursin inhibits VEGF-induced migration and tube formation of HRMECs. A: Human retinal microvascular endothelial cells (HRMECs) were treated with 20 ng/ml vascular endothelial growth factor (VEGF) or 1–50 µM decursin for 24 h. A cell-proliferation assay with [3H]-thymidine was performed. Each value represents the mean (±SD) of three independent experiments. The asterisk indicates a p<0.05. B: HRMECs were treated with 20 ng/ml VEGF or 10 µM decursin for 5 min. Western blot analysis using phospho- (p-)VEGFR-2 and vascular endothelial growth factor receptor-2 (VEGFR-2) antibodies was performed, with β-actin serving as the loading control. Figures were selected as representative data from three independent experiments. Quantitative analysis was performed by measuring the intensity relative to the control. Each value represents means±SEM from three independent experiments. The asterisk indicates a p<0.05. The size of scale bars in figure A, B were 100 µm.

Mentions: To investigate the effect of decursin on VEGF-induced proliferation of retinal endothelial cells, we used 1–50 µM concentrations of decursin on HRMECs. With the treatment of VEGF, proliferation of HRMECs increased 1.98 fold compared with control, which was significantly inhibited by cotreatment with decursin in a dose-dependent manner (Figure 1A).


Decursin inhibits retinal neovascularization via suppression of VEGFR-2 activation.

Kim JH, Kim JH, Lee YM, Ahn EM, Kim KW, Yu YS - Mol. Vis. (2009)

Decursin inhibits VEGF-induced migration and tube formation of HRMECs. A: Human retinal microvascular endothelial cells (HRMECs) were treated with 20 ng/ml vascular endothelial growth factor (VEGF) or 1–50 µM decursin for 24 h. A cell-proliferation assay with [3H]-thymidine was performed. Each value represents the mean (±SD) of three independent experiments. The asterisk indicates a p<0.05. B: HRMECs were treated with 20 ng/ml VEGF or 10 µM decursin for 5 min. Western blot analysis using phospho- (p-)VEGFR-2 and vascular endothelial growth factor receptor-2 (VEGFR-2) antibodies was performed, with β-actin serving as the loading control. Figures were selected as representative data from three independent experiments. Quantitative analysis was performed by measuring the intensity relative to the control. Each value represents means±SEM from three independent experiments. The asterisk indicates a p<0.05. The size of scale bars in figure A, B were 100 µm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2743803&req=5

f1: Decursin inhibits VEGF-induced migration and tube formation of HRMECs. A: Human retinal microvascular endothelial cells (HRMECs) were treated with 20 ng/ml vascular endothelial growth factor (VEGF) or 1–50 µM decursin for 24 h. A cell-proliferation assay with [3H]-thymidine was performed. Each value represents the mean (±SD) of three independent experiments. The asterisk indicates a p<0.05. B: HRMECs were treated with 20 ng/ml VEGF or 10 µM decursin for 5 min. Western blot analysis using phospho- (p-)VEGFR-2 and vascular endothelial growth factor receptor-2 (VEGFR-2) antibodies was performed, with β-actin serving as the loading control. Figures were selected as representative data from three independent experiments. Quantitative analysis was performed by measuring the intensity relative to the control. Each value represents means±SEM from three independent experiments. The asterisk indicates a p<0.05. The size of scale bars in figure A, B were 100 µm.
Mentions: To investigate the effect of decursin on VEGF-induced proliferation of retinal endothelial cells, we used 1–50 µM concentrations of decursin on HRMECs. With the treatment of VEGF, proliferation of HRMECs increased 1.98 fold compared with control, which was significantly inhibited by cotreatment with decursin in a dose-dependent manner (Figure 1A).

Bottom Line: We evaluated the inhibitory effect of decursin on retinal neovascularization.Decursin significantly inhibited VEGF-induced proliferation, migration, and the formation of capillary-like networks of retinal endothelial cells in a dose-dependent manner.Moreover, the upregulation of glial fibrillary acidic protein expression was not detected in Mueller cells.

View Article: PubMed Central - PubMed

Affiliation: Fight against Angiogenesis-Related Blindness (FARB) Laboratory, Department of Ophthalmology, College of Medicine, Seoul National University and Seoul Artificial Eye Center Clinical Research Institute, Seoul National University Hospital, Seoul 151-744, Korea.

ABSTRACT

Purpose: Pathologic angiogenesis in the retina leads to the catastrophic loss of vision. Retinopathy of prematurity (ROP), a vasoproliferative retinopathy, is a leading cause of blindness in children. We evaluated the inhibitory effect of decursin on retinal neovascularization.

Methods: Anti-angiogenic activity of decursin was evaluated by vascular endothelial growth factor (VEGF)-induced proliferation, migration, and in vitro tube formation assay of human retinal microvascular endothelial cells (HRMECs). We also used western blot analysis to assess inhibition of vascular endothelial growth factor receptor-2 (VEGFR-2) phosphorylation by decursin. After intravitreal injection of decursin in a mouse model of ROP, retinal neovascularization was examined by fluorescence angiography and vessel counting in cross-sections. The toxicity of decursin was evaluated through 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in HRMECs as well as histologic and immunohistochemistry examination for glial fibrillary acidic protein in the retina.

Results: Decursin significantly inhibited VEGF-induced proliferation, migration, and the formation of capillary-like networks of retinal endothelial cells in a dose-dependent manner. Decursin inhibited VEGF-induced phosphorylation of VEGFR-2, blocking the VEGFR-2 signaling pathway. When intravitreously injected, decursin dramatically suppressed retinal neovascularization in a mouse model of ROP. Even in a high concentration, decursin never induced any structural or inflammatory changes to cells in retinal or vitreous layers. Moreover, the upregulation of glial fibrillary acidic protein expression was not detected in Mueller cells.

Conclusions: Our data suggest that decursin may be a potent anti-angiogenic agent targeting the VEGFR-2 signaling pathway, which significantly inhibits retinal neovascularization without retinal toxicity and may be applicable in various other vasoproliferative retinopathies as well.

Show MeSH
Related in: MedlinePlus