Regulation of Angiogenesis by Kininogen

激肽原对血管生成的调节

基本信息

批准号：
7746356
负责人：
Keith R. McCrae
金额：
$ 8.22万
依托单位：
CASE WESTERN RESERVE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2008
资助国家：
美国
起止时间：
2008-12-12 至 2010-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7746356
关键词：
Active Sites Angiogenesis Inhibitors Angiostatins Animal Model Animals Antibodies Apoptosis Architecture Arthritis Atherosclerosis Biological Process Blood Vessels Blood flow Bradykinin Bradykinin B2 Receptor Cardiovascular Diseases Cleaved cell Clinical Coagulation Process Collagen Type IV Collagen Type XVIII Development Disease Endostatins Endothelial Cells Extracellular Matrix Family member Female Generations Genes Goals Growth High-Molecular-Weight Kininogen Home environment Human Immunoblotting Inflammation Kallikrein-Kinin System Kininogenase Kininogens Kringles Laboratories Maintenance Malignant Neoplasms Mediating Modeling Morphology Mus Norway Organism Oxidative Stress Oxygen Pathogenesis Pathologic Pathway interactions Phenotype Plasma Plasma Proteins Plasminogen Play Point Mutation Proliferating Protein Fragment Proteins Prothrombin Radioimmunoassay Rattus Regulation Reporting Resistance Retinal Role Screening procedure Stem cells Stimulus Subfamily lentivirinae Techniques Three-dimensional analysis Tissues United States Vascular Diseases Vascular Endothelial Growth Factors Wound Healing angiogenesis density extracellular improved in vivo inhibitor/antagonist insight member mortality mutant neovasculature novel strategies polypeptide public health relevance receptor reproductive response tumor tumor growth

项目摘要

DESCRIPTION (provided by applicant): Over the last decade, an important paradigm has emerged in which conformationally-altered proteins or protein fragments function as endogenous inhibitors of angiogenesis. The parental proteins that give rise to these polypeptides are often members of the family of coagulation and fibrinolytic proteins, or constituents of the extracellular matrix. Recent studies in our laboratory have focused on the mechanisms by which cleaved high molecular weight kininogen (HKa), a member of the intrinsic coagulation pathway, induces apoptosis of proliferating endothelial cells and inhibits angiogenesis. Though HKa inhibits angiogenesis, recent studies in the BN-Ka rat, in which a point mutation in the kininogen gene results in deficient kininogen secretion, suggest that kininogen deficiency results in decreased angiogenesis and tumor growth. This has been attributed to deficient release of bradykinin (BK) from single chain high molecular weight kininogen (HK), leading to diminished activation of stromal BK B2 receptors and subsequent decreases in stromal VEGF secretion. To further investigate this issue, we have deleted one of the two murine kininogen genes (mKng1). Screening of mKng1-/- mice by immunoblotting using several different kininogen antibodies as well as a sensitive BK radioimmunoassay, demonstrates that these animals are completely deficient in kininogen. In direct contradistinction to the BN-Ka rat, preliminary studies in mKng1-/- mice demonstrate that both angiogenesis and tumor growth are increased. We hypothesize that increased angiogenesis in mKng1-/- mice results from deficient generation of anti-angiogenic HKa at sites of active angiogenesis. Since, compared to the rat, the kinin-kallikrein system of the mouse more closely resembles that of the human, we believe that the mKng1-/- mouse provides an important and relevant model for assessing regulation of angiogenesis by kininogen. In this application, we propose to assess the mechanisms underlying the proangiogenic phenotype of mKng1-/- mice through three specific aims. In Specific Aim 1, we will compare tissue morphology, baseline microvascular density, three-dimensional vascular architecture, and the angiogenic response to pathophysiological stimuli in wild type and mKng1-/- mice. These studies will employ newly-developed, automated vessel counting techniques, as well as novel approaches to analysis of three-dimensional vascular morphology. In Specific Aim 2, we will determine whether enhanced angiogenesis in mKng1-/- mice is reversed by replenishment of HK, and whether cleavage of HK to HKa is necessary for reversion to the wild- type phenotype. These studies will employ lentivirus-produced murine HK, as well as a mutant HK resistant to cleavage by kallikrein. In Specific Aim 3, we will assess several important mechanistic issues of potential relevance to the proangiogenic phenotype of mKng1-/- mice, including the levels of circulating endothelial progenitor cells and their ability to home to neovasculature, the intrinsic "angiogenic potential" of mKng1-/- endothelial cells, the role of the uPAR as an "antiangiogenic" HKa receptor, and the importance of oxidative stress to the anti-endothelial cell effects of HKa in vivo. Our observations in mKng1-/- mice establish HK as one of the few genetically-proven endogenous regulators of angiogenesis, and the proposed studies should provide important insight into the mechanisms underlying its activity. PUBLIC HEALTH RELEVANCE: Angiogenesis plays a central role in the pathogenesis of multiple clinical disorders, in particular cancer and cardiovascular disease, the two most common causes of mortality in the United States. For example, angiogenesis is critically involved in the development, progression and metastatic spread of cancer. In cardiovascular disease, angiogenesis may have positive and negative influences. While angiogenesis is essential to the maintenance of blood flow and oxygen delivery to tissues with compromised blood flow due to underlying atherosclerotic vascular disease, angiogenesis may also contribute to the development of atherosclerosis. Angiogenesis also contributes to the progression of many other disorders including arthritis and retinal vascular disease. Kininogen is an abundant plasma protein that is involved in many biological processes, particularly those in which inflammation plays a prominent role. We have demonstrated that the cleaved form of high molecular weight kininogen (HKa) is a potent inhibitor of angiogenesis. We have developed a mouse that lacks the kininogen protein, and shown that angiogenesis and tumor growth are increased in these animals. In this application, we hope to extend these studies, and define the mechanisms by which HKa inhibits angiogenesis in an intact organism. These studies should provide new information concerning these pathways, which are likely to be relevant to the mechanisms of other naturally-occurring angiogenesis inhibitors as well.

描述（由申请人提供）：在过去的十年中，出现了一种重要的范例，其中构象改变的蛋白质或蛋白质片段充当血管生成的内源抑制剂。产生这些多肽的亲本蛋白通常是凝血和纤溶蛋白家族的成员，或者是细胞外基质的成分。我们实验室最近的研究重点是裂解的高分子量激肽原（HKa）（内源性凝血途径的成员）诱导增殖内皮细胞凋亡并抑制血管生成的机制。虽然 HKa 抑制血管生成，但最近对 BN-Ka 大鼠的研究表明，激肽原基因的点突变导致激肽原分泌不足，表明激肽原缺乏会导致血管生成减少和肿瘤生长。这归因于单链高分子量激肽原 (HK) 缓激肽 (BK) 的释放不足，导致基质 BK B2 受体的激活减少以及随后基质 VEGF 分泌的减少。为了进一步研究这个问题，我们删除了两个鼠激肽原基因之一 (mKng1)。使用几种不同的激肽原抗体以及敏感的 BK 放射免疫测定法通过免疫印迹筛选 mKng1-/- 小鼠，表明这些动物完全缺乏激肽原。与 BN-Ka 大鼠直接对比，mKng1-/- 小鼠的初步研究表明血管生成和肿瘤生长均增加。我们假设 mKng1-/- 小鼠中血管生成增加是由于活跃血管生成位点抗血管生成 HKa 生成不足所致。由于与大鼠相比，小鼠的激肽-激肽释放酶系统与人类的激肽-激肽释放酶系统更相似，因此我们相信mKng1-/-小鼠为评估激肽原对血管生成的调节提供了重要且相关的模型。在本申请中，我们建议通过三个具体目标评估 mKng1-/- 小鼠促血管生成表型的潜在机制。在具体目标 1 中，我们将比较野生型和 mKng1-/- 小鼠的组织形态、基线微血管密度、三维血管结构以及对病理生理刺激的血管生成反应。这些研究将采用新开发的自动血管计数技术以及三维血管形态分析的新方法。在具体目标 2 中，我们将确定 mKng1-/- 小鼠中增强的血管生成是否可以通过补充 HK 来逆转，以及 HK 裂解为 HKa 是否是恢复野生型表型所必需的。这些研究将使用慢病毒产生的鼠 HK，以及抗激肽释放酶裂解的突变 HK。在具体目标 3 中，我们将评估与 mKng1-/- 小鼠促血管生成表型潜在相关的几个重要机制问题，包括循环内皮祖细胞的水平及其归巢新血管系统的能力，即 mKng1 内在的“血管生成潜力” -/- 内皮细胞、uPAR 作为“抗血管生成”HKa 受体的作用，以及氧化应激对抗内皮细胞的重要性HKa在体内的作用。我们对 mKng1-/- 小鼠的观察表明 HK 是少数经基因证明的血管生成内源性调节因子之一，拟议的研究应为其活性背后的机制提供重要的见解。公共健康相关性：血管生成在多种临床疾病的发病机制中发挥着核心作用，特别是癌症和心血管疾病，这是美国最常见的两种死亡原因。例如，血管生成与癌症的发生、进展和转移扩散密切相关。在心血管疾病中，血管生成可能产生积极和消极的影响。虽然血管生成对于维持血流和向因潜在动脉粥样硬化血管疾病导致血流受损的组织输送氧气至关重要，但血管生成也可能促进动脉粥样硬化的发展。血管生成还导致许多其他疾病的进展，包括关节炎和视网膜血管疾病。激肽原是一种丰富的血浆蛋白，参与许多生物过程，特别是那些炎症起重要作用的生物过程。我们已经证明，高分子量激肽原 (HKa) 的裂解形式是血管生成的有效抑制剂。我们培育了一种缺乏激肽原蛋白的小鼠，并表明这些动物的血管生成和肿瘤生长增加。在此应用中，我们希望扩展这些研究，并确定 HKa 抑制完整生物体中血管生成的机制。这些研究应该提供有关这些途径的新信息，这些信息也可能与其他天然存在的血管生成抑制剂的机制相关。