MMP‐2 and MMP‐9 synergize in promoting choroidal neovascularization

Abstract

SPECIFIC AIMS Previous studies have shown that the production of gelatinase (MMP-2 and MMP-9) belonging to the matrix metalloproteinase family is increased in human choroidal neovascularization (CNV) occurring during the exudative most aggressive form of age-related macular degeneration (AMD). To more precisely delineate the respective roles of MMP-2 and MMP-9 in choroidal neo-angiogenesis, we investigated their expression and activities in the course of laser-induced murine choroidal neovascularization,. This model was applied to single (MMP-9 KO, MMP-2 KO) or double (MMP-2,9 KO) -deficient mice and to their corresponding wild-type (WT) controls. PRINCIPAL FINDINGS 1. MMP-2 and MMP-9 are produced in lesions associated with choroidal neovascularization Gelatin zymography analysis of ocular posterior segments demonstrated that both MMP-2 and MMP-9 were increasingly produced during the early stages of CNV formation, with the appearance of active forms of MMP-2. In situ zymography revealed a predominant gelatinase activity in the CNV area. 2. Different regulation of MMP-2 and MMP-9 expression RT-PCR evaluation showed that MMP-9 expression was up-regulated during early phases of CNV formation whereas MMP-2 was constitutively expressed without any transcriptional modulation. MT1-MMP mRNA was concomitantly up-regulated, suggesting that the presence of active MMP2 forms was due to the expression and activity of its activator. 3. Severe inhibition of choroidal neovascularization in MMP-2,9 double-deficient mice Fluorescein angiography performed before death (Fig. 1 ⤻ F) showed a significant reduction (PD⤻ ) as compared with each single-deficient mouse (Fig. 1B, C⤻ ) or WT (Fig. 1A⤻ ). Choroidal lesion associated with neovascularization was then quantified by determining the B/C ratio between total lesion thickness (B, maximal height lesion measured from the bottom of the choroid to the top of the neovascular area) to the thickness of adjacent normal choroid (C). A significant reduction of the B/C ratio was observed in MMP-9 (33%), MMP-2 (44%), and MMP-2,9 (56%) -deficient mice vs. their corresponding WT (PE⤻ ). Figure 1. Absence of MMP-2 and MMP-9 prevents the development of experimental choroidal neovascularization. Hematoxylin-eosin staining of a representative area of choroidal neovascularization at the site of laser-induced trauma in control (A) or in mice deficient for MMP-9 (B), MMP-2 (C), and both MMP-2,9 (D). An almost complete absence of neovascularization is visible in mice deficient for both MMP-2 and MMP-9 when vessels were immunostained with anti-mouse PECAM antibody (immunostained in orange with AEC) compared with other conditions, thereby confirming the reduced incidence of neovascularization calculated before death by fluorescein angiography evaluation of the number of leaking laser spots (F). The neovascular reaction was determined with computer-assisted image analysis by evaluating the B/C ratio on day 14 after laser injury of the Bruch’s membrane in single/double-deficient mice and in the corresponding WT controls (E). The neural retina (ret) and choroidal layer (ch) are indicated; neovascular area (arrows). ***P < 0.001; error bars = se. Original magnification, 200×. Download figure Download PowerPoint 4. Fibrinogen/fibrin accumulation in double MMP-2,9-deficient animals Consistent with the modulation of fibrinolysis by matrix metalloproteinase system, we observed by immunohistochemical staining that fibrinogen/fibrin accumulated in double MMP-2,9-deficient animals. In contrast, similar fibrinogen/fibrin deposits were found in WT and single gene-deficient mice. These findings suggest that the absence of both gelatinases impaired fibrinolytic activity in choroidal neovascular membrane. 5. MMP inhibitors decrease the development of CNV In a second approach to assess whether MMPs contribute to CNV development, we first induced endogenous overexpression of TIMP-1 or TIMP-2 by adenoviral-mediated delivery in WT mice. Both TIMP-1 and TIMP-2 overexpression significantly reduced choroidal angiogenesis (PP Figure 2. Schematic representation of putative functions of gelatinase, MT1-MMP, and MMP inhibitors in CNV formation. CNV development is associated with an up-regulation of MMP-9 at a transcriptional level and an activation of pro-MMP-2 by MT1-MMP. This process can be inhibited by physiological (TIMP-1 and TIMP-2) and synthetic inhibitors, impaired in single MMP-deficient mice and abolished in double gelatinase KO mice. Download figure Download PowerPoint In addition, the observation of a synergy between MMP-2 and MMP-9 might be of interest for other pathological conditions associated with angiogenesis such as tumoral development. ¹To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0113fje; doi: 10.1096/fj.03-0113fje