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Jian H. Qi, Sr., Heidi Stoehr, Bela Anand-Apte; Sorsby Fundus Dystrophy S156C-TIMP3 mutation promotes angiogenesis and choroidal neovascularization via an MMP2-dependent mechanism. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3016.
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TIMP3 is a regulator of matrix metalloproteinases (MMPs) and a potent angiogenesis inhibitor. Mutations in the TIMP3 gene cause Sorsby Fundus Dystrophy (SFD), an inherited, early onset macular degenerative disorder. SFD closely resembles age-related macular degeneration (AMD), including the development of choroidal neovascularization (CNV). We have demonstrated previously that expression of SFD-related S156C mutation in human retinal pigment epithelial (RPE) cells reduces MMP inhibition and may promote angiogenesis. The objective of this study was to detertmine if S156C-TIMP3 mutation promotes angiogenesis or CNV via an MMP dependent mechanism.
Methods: Human RPE cells expressing WT and S156C-TIMP3 or empty vector as well as the RPE-choroid tissue of Timp3 S156C/S156C knock-in mice and their corresponding WT littermates were analyzed by reverse zymography and zymography to detect MMP inhibitory activity and gelatinase activity respectively. ELISAs were used to measure MMP2, MMP9, bFGF or VEGF. An in vitro tube formation assay was utilized to analyze angiogenic responses in endothelial cells . In vivo CNV response in mice was quantitated following laser-induced photocoagulation.
S156C-TIMP3 reduced MMP inhibitory activity, increased total MMP2 (pro- and active forms) and bFGF but had no effect on MMP9 and VEGF levels in the conditioned medium (CM) of RPE cells relative to vector controls. In contrast, either WT-TIMP3 or a selective MMP2/MMP9 inhibitor (SB-3CT) inhibited bFGF and VEGF release. Addition of CM from mutant RPE cells to either PAE cells expressing FGF receptor-1 (PAE/FGFR-1) or VEGF receptor-2 (PAE/VEGFR-2) induced increased tube formation in three-dimensional (3D) collagen gels with bFGF and VEGF dependency. Treatment with SB-3CT inhibited mutant cells CM-induced morphogenic effects in PAE/FGFR-1 or PAE/VEGFR-2 cells. Consistent with the results from in vitro experiments, Timp3 S156C/S156C mice showed reduced MMP inhibitory activity and increased active form of MMP2 rather than MMP9 in RPE-choroid tissue, especially after a laser burn. Furthermore, at 2 weeks post-laser induction, the area of CNV at Bruch’s membrane (BM) rupture sites was observed to be significantly larger in mutant mice compared to their WT controls.
S156C-TIMP3 up-regulates RPE cell-mediated angiogenesis by increasing bFGF and VEGF bio-availability and activity via elevated MMP2, and thereby promotes CNV development.
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