Effect of Maspin on Corneal Heme-and lymph- angiogenesis

描述(申请人提供)：角膜是眼睛的屏障，它的透明度是其折射和将光线传输到视网膜所必需的。正常的角膜是无血管的。与角膜血管生成相关的情况包括化学烧伤、慢性接触镜低氧、史蒂文斯-约翰逊综合征、翼状疱疹、移植排斥反应和感染性角膜炎(2-4)。正常的角膜会合成许多抗血管生成的分子，其中之一是Maspin，它是一种非抑制性丝氨酸，调节角膜成纤维细胞以及包括癌细胞在内的其他细胞的黏附、迁移和侵袭。Maspin还抑制角膜微袋模型中血管的生长，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，并开发各种形式的maspin来预防和治疗血管生成。其具体目的是：1)验证maspin通过直接作用于血管和淋巴管内皮细胞以及间接作用于角膜细胞等多种机制改变血红素和淋巴管生成的假说。将测定maspin改变淋巴管生成的能力，并将其与其对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管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)验证Maspin N-末端和/或C-末端区域的磷酸化形式抑制血管生成的假说，这些区域的多肽比完整分子更有效，并开发具有潜在抗血管生成药物的Maspin多肽。该区域的鉴定(S)和maspin抗血管生成特性所需的翻译后修饰将利用由maspin和卵蛋白构建的嵌合蛋白、磷酸化模拟物、磷酸化零型体和maspin多肽。将开发一个血管生成的器官培养模型来测试maspin的形式。3)验证maspin在体内对角膜中的血红素和淋巴管生成起主要调节作用的假设。丢失一个maspin等位基因对血红素和淋巴管生成的影响将在maspin小鼠身上进行测试。将使用抗体进行Maspin重建和耗尽实验。拟议的研究重点是了解血红素和淋巴管生成，这是威胁视力的一个主要公共卫生问题，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大我们对控制角膜血管生成的因素的理解，并将表征这一活动所需的maspin的特性。这些结果将指导maspin突变体和多肽的产生和初步测试，作为治疗新生血管疾病的潜在方式。 与公众健康相关：角膜的一个关键特征是它的透明度，这是光线通过和聚焦到视网膜以实现正常视力所必需的。损伤和疾病会导致角膜中不适当的血管和淋巴管的形成，从而阻碍光线的传输。Maspin是一种抑制角膜血管生长的蛋白质。将进行研究以开发这种蛋白质作为预防角膜血管形成的治疗药物。

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. PUBLIC HEALTH RELEVANCE: A critical feature of the cornea is its transparency, which is required for the passage and focusing of light onto the retina for normal vision. Injury and disease can cause the inappropriate formation of blood and lymph vessels in the cornea that can block light transmission. Maspin is a protein that inhibits vessel growth in the cornea. Studies will be conducted towards developing this protein as a therapeutic for the prevention of corneal vascularization.