Tissue Inhibitor of metalloproteinases-3 (TIMP-3) is a potent matrix-bound angiogenesis inhibitor. Mutations in TIMP-3 cause Sorsby Fundus Dystrophy, a dominant inherited, early onset macular degenerative disease, with choroidal neovascularization causing a loss of vision in the majority of patients. Here we report that expression of S156C TIMP-3 mutation in endothelial cells results in an abnormal localization of the protein, increased glycosylation, decreased matrix metalloproteinase inhibitory activity, and increased vascular endothelial growth factor (VEGF) binding with a consequent increase in VEGF-dependent migration and tube formation. These enhanced signaling events appear to be mediated as a consequence of a post-transcriptionally regulated increase in the expression of membrane-associated VEGFR-2 in endothelial cells of Timp-3 156/156 mutant mice as well as in human Sorsby fundus dystrophy eyes. Understanding the mechanism(s) by which mutant TIMP-3 can induce abnormal neovascularization provides important insight into the pathophysiology of a number of diseases with increased angiogenesis. Tissue inhibitor of metalloproteinases-3 (TIMP-3)2 is a member of the TIMP family of proteins and is an endogenous inhibitor of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase (ADAM), and ADAM with thrombospondin domains (ADAMTS) family of enzymes. TIMP-3 is unique among other members of the TIMP family in that it is tightly bound to the extracellular matrix (ECM). In addition, TIMP-3 is the only TIMP that can inhibit tumor necrosis factor ␣-converting enzyme (TACE/ADAM17) and aggrecanase 1 and 2 (ADAMTS4 and ADAMTS5). Functionally, apart from being a key inhibitor of MMPs (1, 2) and regulating apoptosis in some cells in vitro and in vivo (3-7), 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 (13-20), most of which introduce an unpaired cysteine at the C terminus of the protein. SFD is of considerable interest as it is the only genetic disorder in which choroidal neovascularization occurs in the majority of affected patients (21-23). 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. In this study we have generated endothelial cells (ECs) expressing SFD-related S156C TIMP-3 mutation as a platform and determined how mutant TIMP-3 regulates VEGF signaling via VEGFR-2 during SFD-related angiogenesis. In ad...