Inhibition of choroidal neovascularization by topical application of angiogenesis inhibitor vasostatin

Abstract

Purpose: Choroidal neovascularization (CNV) is the leading cause of blindness in patients with age-related macular degeneration (AMD). This study evaluated the inhibitory effect of vasostatin (VS), an endogenous angiogenesis inhibitor, on CNV.

Methods: Anti-angiogenic activity of VS was evaluated in vitro by migration and tube formation assays in human umbilical vein endothelial cells (HUVECs). CNV lesions were induced in Brown Norway rats by fundus argon laser photocoagulation. Beginning one day after CNV induction, rats were treated with eye drops containing 1 microg/ml VS in PBS buffer for three times daily for 20 days. The extent of CNV was examined by flat mount analysis on day 24 or by fundus fluorescein angiography (FAG) on days 21, 28, 35, and 42, respectively. CNV lesions and choroidal vascularity were evaluated by histological analysis. The spatial distribution of topically applied VS in rat eyes was evaluated by immunoblot analysis.

Results: VS inhibited migration and tube formation in HUVECs. Flat mount analysis revealed that, after laser-induced photocoagulation, topical VS application for 20 days significantly reduced CNV lesions. Moreover, serial FAG analysis indicated that a 20 day VS treatment significantly reduced CNV lesions on all subsequent days. Histological analysis revealed attenuated lesions, intact Bruch's membrane, and reduced choroidal vascularity in VS-treated eyes. Finally immunoblot analysis reveled VS expression in choroids.

Conclusions: Topical VS application suppresses the progression of laser-induced CNV via angiogenesis inhibition and may constitute a therapeutic alternative for excessive neovascularization occurring with ocular diseases.

Figure 1

Anti-angiogenic function of VS in endothelial cells. A: Effect of VS on migration of HUVECs. VS was added in varying doses, and endothelial cells were placed in a Boyden chamber for 6 h and allowed to migrate toward bFGF. Shown are representative photographs of endothelial migration in control and after treatment with 1 μg/ml VS (left). Cell migration was quantified by counting cells from three high power fields and expressed as mean ± SD of triplicates (right). B: Effect of VS on tube formation of HUVECs. Endothelial cells were applied to Matrigel and incubated for 4 h in the presence of VS of varying doses. Representative profiles of the tubular structures in control and 1 μg/ml VS-treated HUVEC are shown (left). Tube formation was quantified by counting the number of rings and expressed as mean±SD from quadruplicates (right). Asterisk (*) represents p<0.05, and double asterisk (**) represents p<0.01.

Figure 2

The experimental scheme of topical VS application for laser-induced CNV. A: Therapeutic efficacy was assessed by flat mount analysis. CNV was induced by laser photocoagulation on day 0 and validated on day 21 by FAG analysis. The animals (n=8) were euthanized on day 24 to measure CNV extent by flat mount analysis. B: In serial FAG analysis, CNV was induced by laser photocoagulation at day 0. From day 1 to 20 after induction, the animals were either left untreated (CNV control group, n=8) or treated with VS eye drops (1 μg/ml in PBS; CNV+VS group, n=8), or PBS vehicle (CNV+PBS group; n=8) three times per day.

Figure 3

Flat mount analysis of choroidal vascularity in rat eyes after topical VS application as determined by FITC-dextran injection. A: FITC-dextran positive blood vessels are presented in choroids of untreated or VS-treated animals. Arrows indicate the laser-induced lesions. B: FITC-dextran labeling CNV in untreated or VS-treated eyes are quantified. Data are summarized as mean±SEM (n=8). Double asterisk (**) represents p<0.01. The scale bar equals 200 μm.

Figure 4

Effect of topical VS application on CNV lesions in rats by FAG. A: Representative photographs of fundus microscopy (top panels) and FAG (bottom panels) indicate the anti-angiogenic effect of topical VS application. Top: the laser spots in rat eyes of different group are shown under fundus microscope. Bottom: FAG analysis of CNV lesion in rat eyes by group are shown on day 28. B: Effect of topical VS application on the area of CNV lesions in rats are evaluated by FAG. Areas of CNV lesions were determined by FAG examination on days 21, 28, 35, and 42 after laser photocoagulation. Data are summarized as mean±SEM (n=8) and are representative of three experiments. Asterisk (*) represents p<0.05, and double asterisk (**) represents p<0.01.

Figure 5

Histological analysis of CNV lesions in rat eyes after topical VS application. After final FAG analysis on day 42, rat eyes were dissected and analyzed by hematoxylin and eosin analysis. VS application resulted in much smaller neovascularization. Arrows indicate the laser-induced CNV lesions. Abbreviations: ganglion cell layer (GL); inner nuclear layer (INL); outer nuclear layer (ONL); choroid (CH). Magnification is 100×.

Figure 6

Immunofluorescence analysis of choroidal vascularity in rat eyes after topical VS application. The choroidal vascularity in PBS-treated or VS-treated eyes was examined by immunofluorescence analysis using anti-vWF. A: Profile of vWF-positive vessels in choroids (C) and retina (R) of PBS-treated or VS-treated eyes are shown. Arrows indicate CNV by little circles in low power filed (arrowheads) and big circles in high power field (larger arrows). B: PBS-treated vWF-positive blood vessels in choroids are nearly twice those of VS-treated eyes. Data are summarized as mean±SEM (n=8). Asterisk (*) indicates p<0.05. The scale bar equals 200 μm.

Figure 7

Immunoblot analysis of the distribution of topically applied VS in rat eyes. Western blot analysis reveals that after 14 days of topical VS application, there is greater 6xHis activity and less β-actin in rat cornea (C) and retinochoroid (RC) compared to PBS-treated rat C and RC.