Abstract
Sorsby's fundus dystrophy (SFD) is an autosomal dominant degenerative disease of the macula caused by mutations in the tissue inhibitor of metalloproteinase-3 (TIMP-3) gene. Choroidal neovascularization is a hallmark of this disease, which closely resembles the exudative form of age-related macular degeneration. However, the mechanism by which TIMP-3 mutations induce the disease phenotype in SFD remains unknown. To address this question we established human retinal pigment epithelial cell lines expressing wild type or S156C (Ser(156) changed to cysteine) mutant TIMP-3. S156C TIMP-3 had reduced matrix metalloproteinase (MMP) inhibitory activity in retinal pigment epithelial cells and resulted in increased secretion and activation of gelatinase A and B. The conditioned medium from these cells induced angiogenesis in "in vivo" chick chorioallantoic membrane assays that could be reversed with recombinant wild type TIMP-3. Our data indicate that the choroidal neovascularization in SFD may be a result of increased MMP activity, which could lead to the stimulation of angiogenesis. These results also suggest the potential therapeutic use of TIMP-3 or synthetic MMP inhibitors in this disease.
Highlights
Sorsby’s fundus dystrophy (SFD),1 a fully penetrant, autosomal dominant, degenerative disease of the macula [1], is manifested by symptoms of night blindness or sudden loss of acuity usually in the third to fourth decades of life due to submacular neovascularization [2,3,4,5]
Like the endogenous tissue inhibitor of metalloproteinase-3 (TIMP-3) in retinal pigment epithelium (RPE) cells, both wild type and mutant forms of TIMP-3 were expressed predominantly in the extracellular matrix (ECM) (Fig. 1, a and b) with a small fraction being secreted into the conditioned medium (Fig. 1, c and d)
Wild type and mutant TIMP-3 proteins were expressed at levels 3–5-fold over that of endogenous TIMP-3 present in ARPE-19 cells
Summary
Cell Culture—ARPE-19 cells (American Type Culture Collection) were maintained in T75 flasks in filter medium consisting of Dulbecco’s modified Eagle’s medium/Ham’s F-12 plus 10% fetal bovine serum (Hyclone). The cells were removed from the culture dishes following a 15-min incubation in Ca2ϩ-, Mg2ϩ-free phosphatebuffered saline (PBS) containing 5 mM EGTA and 1 mM phenylmethylsulfonyl fluoride. Cell Migration Assay—A modified Boyden chamber assay was carried out as described previously [17]. Matrigel and Type I Collagen Gel Invasion Assay—This assay was carried out as described previously [18] with some modifications. Human dermal microvascular endothelial cells were incubated for 72 h in the presence of conditioned medium from RPE cell transfectants. After incubation for 3 days at 37 °C in 3% CO2, CAMs were implanted with 5-mm diameter sterilized gelatin sponges (Gelfoam, Upjohn Co., Kalamazoo, MI) loaded with equal protein amounts of conditioned medium from RPE cell transfectants and photographed on day 3. Responses were graded as Ͼ, ϭ, or Ͻ by independent readers
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