Matrix metalloproteinase-9 contributes to choroidal neovascularization

Abstract

Age-related macular degeneration (AMD) is the primary cause of irreversible photoreceptors loss in adult patients and current therapies are limited. Increased levels of matrix metalloproteinases (MMPs) have been documented in neovascularization of severe ocular pathologies such as AMD and proliferative diabetic retinopathy. We report here that MMP-9 (gelatinase B) expression is induced and temporally regulated in the course of experimental choroidal neovascularization. We used transgenic mice expressing beta-galactosidase reporter gene under the dependence of MMP-9 promoter and RT-PCR analysis on choroidal neovascular structures microdissected from serial sections by laser pressure catapulting to show that MMP-9 expression is up-regulated concomitantly with the appearance of inflammatory cells in the subretinal lesion. In mice deficient in MMP-9 expression the development of choroidal neovascularization induced by laser photocoagulation still occurred, but at a reduced level.

Figure 1.

Temporal pattern of protein and MMP-9 mRNA presence after laser-induced choroidal neovascularization. A—C: Immunolocalization of MMP-9 (red) in ocular frozen sections counterstained with collagen type IV antibody (green) at day 3 (A), day 5 (B) after laser injury of the Bruch’s membrane, and in corneas subjected to thermal injury (C) (positive control). D–F: Mapping of MMP-9 expression using transgene mice expressing β-galactosidase (blue) under the dependence of MMP-9 promoter at day 3 (D) and day 5 (E) in the retina and at day 5 in the cornea (F). White arrows localize the laser impact. The neural retina (ret), retinal pigment epithelium (rpe), choroidal layer (ch), corneal epithelium (ep), corneal stroma (str) and descemet membrane (de) are indicated. Original magnification, ×100.

Figure 2.

Immunolocalization of mononuclear and polymorphonuclear inflammatory cells during the course of choroidal neovascular membrane development after laser injury of the choroid. Mac-1 immunostaining (red) on frozen sections counterstained with bis-benzimide (blue) is absent in the choroid at day 3 (A, B), and appears in the impact at day 5 (C, D). Neutrophils (red) are present at day 3 (E, F), but not at day 5 (data not shown). White arrows localize the laser impact. Original magnifications: A, C, ×100; B, D, E, ×400; F, ×630.

Figure 3.

MMP-2 and MMP-9 expression are differentially modulated during the progression of choroidal neovascular reaction in a murine laser-induced model. Laser pressure catapulting followed by RT-PCR analysis on microdissected choroidal new vessels (I) and on adjacent (control) intact chorioretinal tissue (C) from day 3 to day 40. Molecular weight numbers (bp) are shown at left. The control at day 10 is missing due to limited amount of extracted RNA.

Figure 4.

Zymographic analysis of MMP-2 and MMP-9 in tissues extracted either from posterior eyes segments with laser-induced neovascularization (I, day 3) or intact eyes (C) of three different mice. As positive control, medium conditioned by human HT1080 cells was included. Positions of MMP-2 and MMP-9 are indicated by arrows.

Figure 5.

Histological analysis after laser treatment. Hematoxylin-eosin staining of a representative area of choroidal neovascularization at the site of laser-induced trauma in WT mice (A), and a more restricted reaction in MMP-9^−/− mice (B). Immunofluorescence labeling of new vessels in WT (C) or in MMP-9^−/− mice (D) analyzed 14 days after laser photocoagulation. New vessels were detected with anti-mouse anti-collagen type IV antibody (green) and anti-mouse anti-PECAM antibody (red). The neural retina (ret), retinal pigment epithelium (rpe), and choroidal layer (ch) are indicated and the neovascular area is arrowed. White arrows localize the laser impact. Original magnification, ×200

Figure 6.

Reduced neovascularization in MMP-9^−/− mice. Angiogenesis was quantified by evaluating the B/C ratio as described previously at day 14 after laser injury of the Bruch’s membrane. Results are shown as column scatter; P < 0.001 (unpaired, non-parametric Mann-Whitney).