Role of TIMP-3 in Ocular Neovascularization

描述(申请人提供)：脉络膜新生血管(CNV)是老年性黄斑变性(AMD)患者严重视力丧失的主要原因。新生血管起源于脉络膜毛细血管，通过Bruch膜生长，进入视网膜下色素上皮(RPE)间隙。索尔斯比眼底营养不良(SFD)是一种罕见的、以遗传为主的早发性黄斑变性疾病，是唯一一种在大多数患者中发生脉络膜新生血管的遗传性疾病，引起了人们的极大兴趣。金属蛋白酶组织抑制因子-3(TIMP-3)基因突变可导致SFD。TIMP-3是一种基质金属蛋白酶(MMPs)的抑制剂，由RPE细胞沉积到Bruchs膜(BM)，在那里它是细胞外基质(ECM)的组成部分。我们已经证明，TIMP-3是一种有效的血管生成抑制因子，在这方面的作用不依赖于其对基质金属蛋白酶的抑制。我们还表明，SFD突变体TIMP-3在RPE细胞中的表达减少了对基质金属蛋白酶的抑制，并促进了血管生成。由于CNV是SFD的一个显著特征，我们建议研究野生型和突变型TIMP-3调控新生血管的机制。提出如下模型：在生理条件下，RPE细胞沉积的野生型(Wt)TIMP-3通过阻止VEGF与其受体KDR的结合，有效地抑制脉络膜毛细血管的新生血管侵袭。在SFD的病理性脉络膜新生血管形成过程中，突变型TIMP-3抑制血管生成的能力受损，导致新生血管增加。这项建议的长期目标是了解TIMP-3及其突变调控脉络膜新生血管的机制(S)。利用体外和体内研究，我们的假设将得到以下具体目标的检验。 1.探讨TIMP-3抑制血管生成的分子机制。 A)与蛋白多糖的结合是否在TIMP-3抑制血管生成的能力中发挥作用？ B)TIMP-3基因缺失的小鼠是否表现出视网膜和/或脉络膜血管的异常发育？ C)TIMP-3基因缺失的小鼠对血管内皮生长因子诱导的新生血管更敏感吗？ 2.探讨TIMP-3基因突变在SFD中引起CNV的机制。 A)表达S156C TIMP-3的小鼠对血管内皮生长因子的血管生成反应增强了吗？ B)S156C TIMP-3促进血管内皮生长因子介导的分子机制是什么 血管生成？

DESCRIPTION (provided by applicant): Choroidal neovascularization (CNV) is the major cause of severe vision loss in patients with age-related macular degeneration (AMD). The neovascularization originates from the choriocapillaris and grows through Bruch's membrane, into the sub-retinal pigment epithelial (RPE) space. Sorsby's Fundus Dystrophy (SFD), a rare, dominantly inherited, early onset macular degenerative disease is of considerable interest as it is the only genetic disorder in which choroidal neovascularization occurs in the majority of affected patients. Mutations in the Tissue Inhibitor of Metalloproteinases-3 (TIMP-3) gene cause SFD. TIMP-3, an inhibitor of matrix metalloproteinases (MMPs) is deposited by RPE cells into Bruch's membrane (BM) where it is a component of the extracellular matrix (ECM). We have demonstrated that TIMP-3 is a potent inhibitor of angiogenesis and functions independent of its MMP inhibition in this respect. We have also shown that expression of SFD mutant TIMP-3 in RPE cells reduces MMP inhibition and promotes angiogenesis. Since CNV is a prominent feature of SFD, we propose to study the mechanisms by which wild type and mutant TIMP-3 regulate neovascularization. The following model is proposed: Under physiological conditions, wild type (wt) TIMP-3 deposited by RPE cells into Bruch's membrane efficiently inhibits neovascular invasion from the choriocapillaris by preventing the binding of VEGF to its receptor KDR. During pathological choroidal neovascularization as seen in SFD, mutant TIMP-3 is compromised in its angiostatic ability and results in increased neovascularization. The long term goal of this proposal is to gain an understanding of the mechanism(s) by which TIMP-3 and its mutations regulate choroidal neovascularization. Using both in vitro and in vivo studies our hypothesis will be tested with the following Specific Aims. 1. To determine the molecular mechanism by which TIMP-3 inhibits angiogenesis. a) Does binding to proteoglycan play a role in the ability of TIMP-3 to inhibit angiogenesis? b) Do TIMP-3 null mice demonstrate abnormal development of retinal and/or choroidal vasculature? c) Are TIMP-3 null mice more susceptible to VEGF induced CNV? 2. To determine the mechanism by which TIMP-3 mutations cause CNV in SFD. a) Do mice expressing S156C TIMP-3 show increased angiogenic responses to VEGF? b) What are the molecular mechanisms by which S156C TIMP-3 accentuates VEGF mediated angiogenesis?