Antiangiogenic effects of topically administered multiple kinase inhibitor, motesanib (AMG 706), on experimental choroidal neovascularization in mice

Abstract

Purpose: To investigate the effect of topical motesanib, an inhibitor of receptor tyrosine kinase, on experimental choroidal neovascularization (CNV).

Methods: CNV was induced in 46 nine-week-old male C57BL/6 mice using fundus laser photocoagulation. The right eye of each mouse was treated with motesanib eye drop (4 times daily) and the left eye with vehicle eye drop (4 times daily) for 14 days. To evaluate changes in the CNV lesions, fluorescein angiography, immunofluorescence staining with CD34, and histological examinations were performed 14 days after CNV induction. The expression of phosphorylated extracellular signal-regulated kinase (ERK1/2) in choroidal tissues was determined using western blot analysis to demonstrate the inhibitory effect of topically administered motesanib on intracellular signaling pathways involved in CNV development.

Results: Fluorescein angiography showed that fluorescence leakage in eyes treated with topical motesanib was significantly less than in mice treated with vehicle (P=0.01). On immunofluorescence staining, the CD34-labeled area was smaller in topical motesanib-treated eyes (P<0.001). The expression level of phosphorylated ERK1/2 relative to that of total ERK1/2 decreased in eyes treated with topical motesanib compared with eyes treated with vehicle.

Conclusion: Topical motesanib significantly reduced laser-induced CNV in the experimental mouse model.

FIG. 1.

Fluorescein angiograms (n=20, n: the number of animals in each group) displayed leakage of fluorescein in the photocoagulated lesions of (A) vehicle-treated and (B) topical motesanib-treated eyes 14 days after laser injury. (C) The signal intensities were measured and analyzed. Signal intensities were presented from 0 (darkest) to 1 (brightest) for each pixel of the image in the choroidal neovascularization (CNV) lesions using an arbitrary unit. As a reference, the intensity within a nonphotocoagulated capillary area (arrow) was defined as 0 and the intensity at the major branch of the retinal vein (arrowhead) was defined as 1. Topically administered motesanib significantly reduced the fluorescein leakage of CNV lesions (*P=0.01).

FIG. 2.

Immunofluorescence staining of CNV lesions 14 days after laser photocoagulation. Choroidal flat mounts were fluorescently labeled with F-actin-specific marker phalloidin (green channel), endothelial cell marker CD34 (red channel), and nuclear marker DAPI (blue channel). CNV lesions in topical vehicle-treated eye (A, C, E and G) and topical motesanib-treated eye (B, D, F and H). The scale bar represents 100 μm. Color images available online at www.liebertpub.com/jop

FIG. 3.

The size of endothelial cell marker CD34-labeled area of topical motesanib-treated eye was smaller than that of the vehicle-treated eye (*P=0.001).

FIG. 4.

(A) Phosphorylated extracellular signal-regulated kinase (p-ERK1/2) expression in retinal pigment epithelium/choroid tissue of vehicle-treated left eye and topical motesanib (5 mg/mL)–treated right eye 7 days after laser injury. (B) Densitometric analysis showed that the expression of p-ERK1/2 was suppressed by topical motesanib treatment. V: vehicle-treated eye; T: motesanib-treated eye. *P=0.05.

FIG. 5.

Effect of topical motesanib on CNV development 14 days after laser induction of CNV. Light micrographs of hematoxylin–eosin-stained sections of the CNV lesions in (A) vehicle-treated and (B) topical motesanib-treated eyes. Note that the thickness of CNV (arrows) was decreased in the topical motesanib-treated eye. The scale bar represents 100 μm. Color images available online at www.liebertpub.com/jop