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ANGIOCIDIN, A NEW ANGIOGENESIS INHIBITOR

血管抑制素，一种新的血管生成抑制剂

基本信息

批准号：
6765993
负责人：
GEORGE Paul TUSZYNSKI
金额：
$ 35.71万
依托单位：
TEMPLE UNIV OF THE COMMONWEALTH
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-07-01 至 2005-12-31
项目状态：
已结题

项目摘要

We have cloned a novel high affinity thrombospondin-1 (TSP-1) binding protein from a prostate cancer cell library. The protein, termed angiocidin, inhibited endothelial cell viability and tube formation in vitro and prevented the growth of tumors in vivo. When injected intravenously into mice bearing Lewis Lung carcinoma, the protein inhibited the growth of the tumor by more than 500% as compared to the buffer control. As shown by immunohistochemical staining of human breast tumors angiocidin localized to malignant ductal epithelium while no significant staining of epithelium in normal and benign tissues was observed. In this proposal we will test the hypothesis that angiocidin exerts its anti-angiogenic activity by competing with TSP-1, its high affinity ligand, and regulating expression of other endothelial proteins important in angiogenesis. We will establish which steps in the angiogenic process are inhibited by angiocidin, what structural determinants of angiocidin mediate this activity, and what other proteins may be binding and regulating angiocidin activity. Our goal is to understand how angiocidin blocks angiogenesis. Cur long range goal is to develop angiocidin as a treatment for cancer. These goals will be accomplished by the following aims. In aim 1, we will establish the capacity of angiocidin to modulate the following TSP-1 dependent biological steps important in angiogenesis: a) endothelial cell adhesion to extracellular matrix b) growth factor dependent endothelial cell proliferation c) growth factor dependent endothelial cell invasion. In aim 2 we will map the structural domains of angiocidin that mediate its anti- angiogenic activity and TSP-1 binding activity with synthetic peptides and monoclonal antibodies whose binding epitopes will be mapped using peptide phage display. In aim 3 we will determine the molecular basis of the anti-angiogenic activity of angiocidin by establishing that endothelial cells bind angiocidin through cell surface receptors, which will be identified by affinity chromatography and molecular cloning. To further unravel the anti-angiogenic activity of angiocidin, genes regulated by angiocidin will be identified by cDNA expression array analysis. Finally in aim 4 the in vivo activity of angiocidin in preventing tumor growth as well as its in vivo distribution will be evaluated in a syngeneic mouse and orthotopic xenograft mouse model of tumor growth. These models will be used to show that injected angiocidin as well as angiocidin produced by transfected tumor cells decreases tumor microvessel density. The role of TSP-1 in the activity of angiocidin will be further evaluated in tumor bearing TSP-1 null mice. The experiments outlined above should advance the field of angiogenesis research by filling the gap in our understanding of how angiogenesis is regulated by molecules like angiocidin and its ligand TSP- 1. Finally, these experiments should provide the basis for the development of angiocidin as an anti-cancer therapeutic.

我们从前列腺癌细胞文库中克隆了一个新的高亲和力凝血酶敏感蛋白-1(TSP-1)结合蛋白。这种蛋白质被称为血管杀菌素，在体外抑制血管内皮细胞的活性和管子的形成，并在体内阻止肿瘤的生长。当静脉注射到荷Lewis肺癌的小鼠体内时，与缓冲液对照组相比，该蛋白对肿瘤的生长抑制了500%以上。免疫组织化学染色显示，血管抑素定位于恶性导管上皮细胞，而在正常组织和良性组织中未见明显染色。在这个提案中，我们将验证这样的假设，即血管生成抑制素通过与其高亲和力配体TSP-1竞争，并调节在血管生成中重要的其他内皮蛋白的表达来发挥其抗血管生成活性。我们将确定血管生成过程中的哪些步骤被血管生成蛋白抑制，血管生成蛋白的哪些结构决定因素介导这种活性，以及哪些其他蛋白可能结合和调节血管生成蛋白的活性。我们的目标是了解血管杀菌素是如何阻止血管生成的。Cur的长期目标是开发血管杀菌素作为癌症的治疗方法。这些目标将通过以下目标实现。在目标1中，我们将确定Angioidin调节以下TSP-1依赖的生物学步骤在血管生成中的重要作用：a)内皮细胞与细胞外基质的黏附；b)依赖生长因子的内皮细胞增殖；c)依赖生长因子的内皮细胞侵袭。在目标2中，我们将用合成肽和单抗来定位介导其抗血管生成活性和TSP-1结合活性的血管杀菌素的结构域，其结合表位将用肽噬菌体展示来绘制。在目标3中，我们将通过建立内皮细胞通过细胞表面受体与血管生成素结合来确定血管生成素抗血管生成活性的分子基础，这将通过亲和层析和分子克隆来鉴定。为了进一步揭示血管生成蛋白的抗血管生成活性，将通过基因表达阵列分析来鉴定血管生成蛋白调控的基因。最后，在目标4中，将在同基因小鼠和原位异种移植小鼠肿瘤生长模型中评价血管杀菌素的体内抑制肿瘤生长的活性及其在体内的分布。这些模型将被用来显示注射血管杀菌素以及由转基因肿瘤细胞产生的血管杀菌素降低肿瘤微血管密度。TSP-1在血管抑素活性中的作用将在荷瘤TSP-1基因缺失的小鼠中进一步评估。上述实验将填补我们对血管生成如何受血管生成蛋白及其配体TSP-1等分子调控的空白，从而推动血管生成研究领域的发展。最后，这些实验将为血管生成蛋白作为抗癌治疗药物的开发提供基础。