Abstract
Abstract: :
Purpose: Sorsby fundus dystrophy (SFD) is a dominantly inherited condition characterized by the development of choroidal neovascularization, subretinal hemorrhages and changes consistent with disciform degeneration. Mutations in the Tissue Inhibitor of Metalloproteinases–3 (TIMP–3) gene, all of which introduce an extra cysteine residue into exon 5, cause Sorsby Fundus Dystrophy (SFD). TIMP–3, a regulator of matrix metalloproteinases (MMPs) is deposited by retinal pigment epithelial (RPE) cells into Bruch's membrane (BM) where it is a component of the extracellular matrix. In this study we set out to elucidate the mechanism by which TIMP–3 mutations induce the angiogenic phenotype. Methods: We have introduced TIMP–3 (WT or S156C) into porcine aortic endothelial cells expressing VEGFR–2. Subcellular localization of WT and mutant protein was analyzed by western blot analysis. MMP inhibition was examined by reverse zymography and caspase activity was quantitated for apoptosis. In vitro, VEGF induced proliferation and migration of endothelial cells were analyzed by Coulter particle counting and modified Boyden chamber assays respectively. Results: S156C TIMP–3 is present in the extracellular matrix (ECM) but unlike WT TIMP–3 a large proportion of the protein is secreted into the conditioned medium (2 fold over ECM). In addition, there appears to be an accumulation of the S156C TIMP–3 in the lysates of cells (approximately 3 fold more than that seen with WT TIMP–3). Reverse zymography determined that S156C TIMP–3 is an inefficient MMP inhibitor in endothelial cells. However, like the WT TIMP–3, mutant TIMP–3 can still increase the caspase activity and promote apoptosis. Mutant TIMP–3 can also block the binding of VEGF to VEGFR–2 and inhibit downstream signaling and cell migration and proliferation in response to VEGF. Conclusions: Our data indicate that expression of SFD mutant S156C TIMP–3 in endothelial cells results in attenuated MMP inhibition. This however has no effect on VEGF mediated angiogenic responses, which confirms that angiogenesis inhibition by TIMP–3 occurs via an MMP independent mechanism.
Keywords: neovascularization • retina • pathobiology