Effect of Maspin on Corneal Heme-and lymph- angiogenesis

描述（由申请人提供）：角膜作为眼睛的屏障，其透明性是其功能所必需的，以将光吸收并传输到视网膜。正常的角膜是无血管的。与角膜血管生成相关的病症的实例包括化学烧伤、慢性接触透镜缺氧、Stevens-Johnson综合征、翼状胬肉、移植排斥和感染性角膜炎（2-4）。正常角膜合成许多抗血管生成分子，其中之一是maspin，一种非抑制性丝氨酸蛋白酶抑制剂，其调节角膜成纤维细胞以及其他细胞类型（包括癌细胞）的粘附、迁移和侵袭。Maspin还抑制角膜微囊模型中的血管向内生长，并抑制微血管内皮细胞的管形成、增殖和迁移。本项目的总体目标是了解maspin抑制角膜血管生成的机制，并开发用于预防和治疗血管生成的maspin形式。 具体目标是：1）检验maspin通过多种机制改变血红素和淋巴管生成的假设，所述机制涉及对血管和淋巴内皮细胞的直接作用和对角膜细胞的间接作用。将测定maspin改变淋巴管生成的能力，并将其与其抗血红素血管生成的活性进行比较。将确定maspin对角膜上皮和基质成纤维细胞以及血红素和淋巴内皮细胞合成的促血管生成蛋白和抗血管生成蛋白水平的影响。 2)检验maspin的N-末端和/或C-末端区域的磷酸化形式抑制血管生成的假设，这些区域的肽比完整分子更有效，并开发具有抗血管生成药物潜力的maspin肽。maspin的抗血管生成特性所需的maspin的区域和翻译后修饰的鉴定将利用由maspin和卵清蛋白、磷酸化模拟物、磷酸化无效形式和maspin肽构建的嵌合蛋白。将开发血管生成的器官培养模型以测试maspin形式。 3)检验maspin在角膜中血红素和淋巴血管生成的体内主要调节作用的假设。将使用maspin小鼠测试一个maspin等位基因缺失对血红素和淋巴管生成的影响。将使用抗体进行Maspin重建和消除实验。 拟议的研究重点是了解血红素和淋巴管生成，这是一个威胁视力的主要公共卫生问题，旨在产生新的治疗方法。这些研究还将通过鉴定抗血管生成分子maspin改变血管生成的机制来扩展我们对控制角膜血管生成的因素的理解，并将表征这种活性所需的maspin的特性。这些结果将指导maspin突变体和肽作为治疗新生血管疾病的潜在方式的产生和初步测试。

DESCRIPTION (provided by applicant): The cornea serves as a barrier for the eye and its transparency is required for its function to refract and transmit light to the retina. The normal cornea is avascular. Examples of conditions associated with corneal angiogenesis include chemical burns, chronic contact lens hypoxia, Stevens-Johnson syndrome, pterygia, graft rejection and infectious keratitis (2-4). The normal cornea synthesizes a number of anti-angiogenic molecules, one of which is maspin, a non-inhibitory Serpin that regulates adhesion, migration and invasion of corneal fibroblasts as well as other cell types, including carcinoma cells. Maspin also inhibits in-growth of vessels in the corneal micropocket model and inhibits tube formation, proliferation and migration of microvascular endothelial cells. The overall goal of this project is to understand the mechanism of maspin inhibition of corneal angiogenesis and to develop forms of maspin for the prevention and treatment of angiogenesis. The Specific Objectives are: 1) To test the hypothesis that maspin alters heme and lymph-angiogenesis by multiple mechanisms involving both direct effects on vascular and lymph endothelial cells and indirect effects on corneal cells. The ability of maspin to alter lymph-angiogenesis will be determined and compared with its activity against heme-angiogenesis. The effect of maspin on the levels of pro-and anti-angiogenic proteins synthesized by corneal epithelial and stromal fibroblast cells and heme- and lymph-endothelial cells will be determined. 2) To test the hypothesis that the phosphorylated form of the N-terminal and/or C-terminal region of maspin inhibits angiogenesis, with peptides of these regions more potent than the intact molecule, and to develop maspin peptides that have potential as anti-angiogenic drugs. Identification of the region(s) and post-translational modifications of maspin required for its anti-angiogenic properties will utilize chimeric proteins constructed from maspin and ovalbumin, phosphorylation mimics, phosphorylation null forms and maspin peptides. An organ culture model of angiogenesis will be developed to test the maspin forms. 3) To test the hypothesis that maspin plays a major in vivo regulatory role of heme- and lymph- angiogenesis in the cornea. The effect of loss of one maspin allele on heme- and lymph-angiogenesis will be tested using maspin mice. Maspin reconstitution and depletion experiments with antibodies will be used. The proposed studies focus on understanding heme- and lymph-angiogenesis, a major public health issue that threatens vision, and aims to generate new treatment methods. The studies will also extend our understanding of factors that control corneal angiogenesis through identification of the mechanisms by which the anti-angiogenic molecule maspin alters angiogenesis and will characterize the properties of maspin required for this activity. The results will guide the generation and initial testing of maspin mutants and peptides as potential modalities for the treatment of neovascular diseases.