Inhibition of choroidal neovascularization by homoisoflavanone, a new angiogenesis inhibitor

Abstract

Purpose: Age-related macular degeneration (AMD) is the leading cause of blindness in elderly. The detailed mechanism of choroidal neovascularization (CNV) leads to severe vision loss in patients with AMD. This study was undertaken to evaluate the inhibitory effect of homoisoflavanone on CNV.

Methods: Antiangiogenic activity of homoisoflavanone was evaluated by in vitro tube formation assay of human umbilical vein endothelial cells (HUVECs) and cell migration assay of HUVECs., Homoisoflavanone or PBS was injected intravitreously into a mouse model of laser-photocoagulation-induced CNV. Fluorescein angiography and vessel counting in cross sections were employed to examine CNV lesions. The toxicity of homoisoflavanone was evaluated through 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay (MTT) assay in HUVECs as well as histological examination and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining in the retina.

Results: Homoisoflavanone effectively inhibited in vitro tube formation and cell migration of HUVECs. Interestingly, homoisoflavanone significantly reduced CNV and its leakage in a mouse model of laser-photocoagulation-induced CNV. In addition, homoisoflavanone showed no effect on cell viability of HUVECs and no retinal toxicity in a concentration range of 1-10 microM.

Conclusions: Our data suggest that homoisoflavanone is a potent inhibitor of CNV and may be applied in the treatment of other vasoproliferative retinopathies and tumor.

Figure 1

Effect of homoisoflavanone on fibroblast growth factor-2 and human umbilical vein endothelial cells. A: Each figure is representative of three independent experiments. B: Basal tube formation of human umbilical vein endothelial cells (HUVECs) left in serum-free media was normalized to 100% respectively. Each value represents means ± SEM from three independent experiments (*p<0.05).

Figure 2

Effect of homoisoflavanone on fibroblast growth factor-2-induced cell migration of human umbilical vein endothelial cells. A: Each figure is representative of three independent experiments. B: The basal migration of human umbilical vein endothelial cells (HUVECs) that were left in serum-free media was normalized to 100% respectively. Each value represents means ± SEM from three independent experiments (*p<0.05).

Figure 3

Effect of homoisoflavanone on laser-photocoagulation-induced choroidal neovascularization. Choroidal neovascularization (CNV) in control and homoisoflavanone-treated mice was evaluated by fluorescein angiography using 500,000 molecular weight fluorescein-conjugated dextran. Wholemount preparation from control (A) and 1 µM homoisoflavanone-treated (B) mice subjected to laser-photocoagulation-induced CNV was performed after 1 h perfusion of fluorescein-conjugated dextran, respectively. Circles indicate CNV in the laser-photocoagulation site. Hematoxylin-stained cross-sections were prepared from control (C) and 1 μM homoisoflavanone-treated (D) mice subjected to laser, respectively. Arrows show the new vessels growing from choroidal vessels. E: To quantify CNV, we counted vessels from subretinal fibrovascular membrane. Data in each column are the mean ± standard deviation values from 100 sites of 25 mice (*p<0.05). Scale bars in C and D equal 50 µm.

Figure 4

Effect of homoisoflavanone on the viability of human umbilical vein endothelial cells. Various concentrations of homoisoflavanone (1-10 μM) were treated on human umbilical vein endothelial cells (HUVECs), and cells were incubated for two days. Cell viability was measured by MTT assay. Each value represents means ± SEM from three independent experiments (*p>0.05).

Figure 5

Retinal toxicity of homoisoflavanone. Ten μM homoisoflavanone was intravitreously injected, and the globes were enucleated three days after treatment. The retina was normal without any inflammatory cells in the vitreous, retina, or choroid. Compared to control, TUNEL-positive cells were not increased with homoisoflavanone injection. Abbreviations: ganglion cell layer (GCL); inner nuclear layer (INL); outer nuclear layer (ONL). Scale bars equal 50 µm.