Expression of Sorsby's Fundus Dystrophy Mutations in Human Retinal Pigment Epithelial Cells Reduces Matrix Metalloproteinase Inhibition and May Promote Angiogenesis

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.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 (2-5). Clinically, early mid-peripheral drusen and color vision deficits are found (4, 5). SFD is a relatively rare disease, but it has generated significant interest because it closely resembles the exudative or "wet" form of age-related macular degeneration, the most common cause of blindness in the elderly population of the western world. SFD is characterized by accumulation of extracellular deposits (drusen) in Bruch's membrane, the five-layered sheet of connective tissue that separates the retinal pigment epithelium (RPE) from the choriocapillaris (6). The predominant histopathological feature in the eyes of a 63-year-old patient with SFD was a confluent 30-m thick, lipid-containing amorphous deposit found between the basement membrane of the RPE and the inner collagenous layer of Bruch's membrane (6). This is distinct from the basal linear deposits seen in age-related macular degeneration that consist of filamentous fine granular material and may represent a thickened basement membrane of the RPE (6). The subretinal deposits in both SFD and age-related macular degeneration have been shown to be rich in tissue inhibitor of metalloproteinase-3 (TIMP-3) (7,8). A serious complication of SFD is the invasion of the thickened Bruch's membrane by newly formed, thin-walled vessels derived from the choriocapillaris. These vessels grow into the subretinal space causing exudative detachment of the RPE and loss of photoreceptors (5). SFD has been linked with mutations in the TIMP-3 gene (10 -12) with five different missense mutations and a splice site mutation having been identi...

S156C Mutation in Tissue Inhibitor of Metalloproteinases-3 Induces Increased Angiogenesis

“…1C), which suggests that the 28-kDa protein is a glycosylated variant of mutant TIMP-3. The spatial expression of mutant TIMP-3 and its molecular mass species in endothelial cells are distinct from those observed in RPE cells (10,26), where the mutant protein is expressed in the ECM like its wildtype counterpart. The reason for this discrepancy is likely because of unique distributions and expressions in different cell types.…”

“…Materials-Porcine aortic endothelial (PAE and PAE KDR ) cells were cultured in Ham's F-12/Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT), 50 unit/ml penicillin, and 50 g/ml streptomycin as described previously (10). Recombinant human VEGF was a kind gift from Genentech.…”

“…SFD is of considerable interest as it is the only genetic disorder in which choroidal neovascularization occurs in the majority of affected patients (21)(22)(23). SFD-TIMP-3 protein accumulates in deposits in Bruch membrane (24) and shows pro-apoptotic activity (25) and reduced MMP inhibitory activity (10). Whether SFD-TIMP-3 affects VEGFR-2-mediated VEGF signaling specific to angiogenesis is unknown.…”

“…Functionally, apart from being a key inhibitor of MMPs (1,2) and regulating apoptosis in some cells in vitro and in vivo (3)(4)(5)(6)(7), TIMP-3 has been demonstrated to be a potent inhibitor of angiogenesis through its ability to block the binding of VEGF to its receptor VEGFR-2 (also known as KDR and FLK-1), affecting subsequent receptor downstream signaling (8). TIMP-3 protein is produced constitutively by the retina pigment epithelium (RPE) and choroidal endothelial cells (9,10) in the eye and is a component of the normal Bruch membrane (11). Sorsby fundus dystrophy (SFD) (12), a dominantly inherited, degenerative disease of the macula, is caused by specific mutations in the TIMP-3 gene (13)(14)(15)(16)(17)(18)(19)(20), most of which introduce an unpaired cysteine at the C terminus of the protein.…”

“…Reverse Zymography-Conditioned medium, cell lysates, and ECM fractions from transfected cells were prepared as described previously (10). Equal amounts of protein were loaded onto a 12% gel with 1 mg/ml gelatin plus RPE cell-conditioned media, as a source of MMPs, for reverse zymography and processed as described previously (2).…”