Biological effects of herbal molecules in ocular neovascularization in vitro and in vivo.

摘要

Angiogenesis is a process of new blood vessels sprouting from the pre-existing vasculature, and mediated by multiple angiogenic and anti-angiogenic factors. Disturbance of the balance often leads to development of neovascular diseases. Neovascularization affecting the eye is a common cause of visual impairment and even blindness, particularly when corneal or choroidal neovascularization (NV) is involved. While there are effective treatment modes for ocular neovascularization, they are expensive and only inhibit disease progress. Since herbal medicine has been applied for anti-angiogenesis and anti-carcinogenesis therapies, we investigate the anti-angiogenic effect of selected herbal molecules: isoliquiritigenin (ISL), a flavonoid from licorice; epigallocatechin gallate (EGCG), a polyphenol from green tea; and resveratrol (Rst), a polyphenol phytoalexin derived from grapes.;This thesis contains two major parts. The first in vitro cell-based analysis investigated the toxicity of these herbal chemicals and their effect on endothelial cell growth and migration. The expression profile of vascular endothelial growth factor (VEGF) signaling cascade events, including Akt and focal adhesion kinase (FAK) activation, VEGF, pigment epithelium-derived factor (PEDF) and matrix metalloproteinases (MMPs) were examined by Western blotting. Then three in vivo models were established to study the effect of these herbal chemicals on angiogenesis. They were (1) developmental angiogenesis in chick chorioallantoic membrane (CAM), (2) pathological angiogenesis in silver nitrate cauterization-induced corneal neovascularization in BALB/c mice and, (3) laser photocoagulation-induced choroidal neovascularization in C57BL/6 mice. Changes of vascularization were determined by qualification of vessel number changes on the edge of gelatin sponge in 24 hours (chick CAM assay), measurement of vascularized area, live imaging of vessel leakage (fundus fluorescence angiography, FFA) and immunochemistry using antibodies specific for endothelial cells (corneal & choroidal NV assays) respectively.;Results showed that sub-toxic levels of ISL (10 µM), EGCG (50 µM) and Rst (10µM) effectively suppressed endothelial cell proliferation and migration in the scratch-wound assay. Treatment with ISL was found to significantly up-regulate PEDF, which is known as a potent angiostatic factor. EGCG and Rst downregulate VEGF signaling cascade by suppressing Akt and FAK activation and affecting MMP-2, MMP-9 expression. In vivo angiogenesis assays further showed the suppressive effect of ISL, EGCG and Rst on neovascularization in three different animal models. Application of ISL at 1 µM showed the suppressive effect on chick CAM assay, corneal NV and choroidal NV assays consistently, the most effective dosage was close to 10 µM. EGCG at 1 µM showed the effect to reduce chick CAM vessel formation and corneal NV, and at 10 µM (the lowest tested concentration) to suppress choroidal NV in mice. Variable effects were observed in Rst treatment. Rst at 10 µM prohibited vessel growth in chick CAM, and 1 µM suppressed corneal NV formation and 2 µM deterred choroidal NV development.;In conclusion, by in vitro and in vivo studies, we showed that ISL, EGCG and Rst contributed to anti-angiogenesis via different biological mechanisms. We propose that these three herbal molecules (ISL, EGCG and Rst) are candidate anti-angiogenic agents for the treatment of ocular angiogenesis diseases. Their distribution profiles and pharmacokinetic properties should be investigated.