Role of TIMP-3 in Ocular Neovascularization

描述（由申请人提供）：脉络膜新生血管（CNV）是年龄相关性黄斑变性（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，并通过布鲁赫膜生长到视网膜色素上皮（RPE）下空间。Sorsby眼底营养不良（SFD）是一种罕见的显性遗传性早发性黄斑变性疾病，由于它是大多数受影响患者中发生脉络膜新生血管的唯一遗传性疾病，因此受到相当大的关注。金属蛋白酶组织抑制剂-3（TIMP-3）基因突变导致SFD。基质金属蛋白酶（MMP）的抑制剂TIMP-3通过RPE细胞沉积到布鲁赫膜（BM）中，在那里它是细胞外基质（ECM）的组分。我们已经证明，TIMP-3是一种有效的血管生成抑制剂，在这方面，其功能独立于MMP抑制。我们还表明，SFD突变体TIMP-3在RPE细胞中的表达降低MMP抑制并促进血管生成。由于CNV是SFD的突出特征，我们建议研究野生型和突变型TIMP-3调节新生血管形成的机制。提出了以下模型：在生理条件下，由RPE细胞沉积到布鲁赫膜中的野生型（wt）TIMP-3通过阻止VEGF与其受体KDR的结合而有效地抑制来自脉络膜毛细血管的新生血管侵袭。在SFD中所见的病理性脉络膜新生血管形成过程中，突变型TIMP-3的血管抑制能力受损，导致新生血管形成增加。该建议的长期目标是了解TIMP-3及其突变调节脉络膜新生血管形成的机制。使用体外和体内研究，我们的假设将通过以下特定目的进行检验。 1.确定TIMP-3抑制血管生成的分子机制。 a）与蛋白聚糖的结合是否在TIMP-3抑制血管生成的能力中起作用？ B）TIMP-3缺失小鼠是否表现出视网膜和/或脉络膜脉管系统的异常发育？ c）TIMP-3缺失小鼠对VEGF诱导的CNV更敏感吗？ 2.确定TIMP-3突变导致SFD中CNV的机制。 a）表达S156 C TIMP-3的小鼠是否显示对VEGF的血管生成反应增加？ B）S156 C TIMP-3增强VEGF介导的VEGF表达的分子机制是什么？ 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?