Regulation of Angiogenesis by Kininogen

激肽原对血管生成的调节

• 批准号: 8033860
• 负责人: Keith R. McCrae
• 金额: $ 32.69万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-12-12 至 2013-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8033860
• 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

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作为血管生成的内源性抑制剂起作用。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，或细胞外基质的成分。我们实验室最近的研究集中在内源性凝血途径的一员，裂解的高分子量激肽原（HKa）诱导增殖内皮细胞凋亡和抑制血管生成的机制。虽然HKa抑制血管生成，但最近在BN-Ka大鼠中的研究（其中激肽原基因中的点突变导致激肽原分泌缺陷）表明，激肽原缺陷导致血管生成和肿瘤生长减少。这归因于从单链高分子量激肽原（HK）释放缓激肽（BK）的不足，导致基质BK B2受体的活化减少和随后基质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（mKng 1）。使用几种不同的激肽原抗体通过免疫印迹以及灵敏的BK放射免疫分析筛选mKng 1-/-小鼠，表明这些动物完全缺乏激肽原。与BN-Ka大鼠直接相反，mKng 1-/-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mKng 1-/-小鼠中血管生成的增加是由于在活跃血管生成的部位缺乏抗血管生成HKA。由于与大鼠相比，小鼠的激肽-激肽释放酶系统更接近于人的激肽-激肽释放酶系统，因此我们认为mKng 1-/-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mKng 1-/-小鼠促血管生成表型的机制。在特定目标1中，我们将比较野生型和mKng 1-/-小鼠的组织形态学、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化血管计数技术，以及分析三维血管形态的新方法。在特异性目标2中，我们将确定mKng 1-/-小鼠中增强的血管生成是否通过补充HK逆转，以及HK裂解为HKa是否是逆转为野生型表型所必需的。这些研究将采用慢病毒产生的小鼠HK，以及突变HK抗激肽释放酶裂解。在具体目标3中，我们将评估与mKng 1-/-小鼠的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至新血管的能力，mKng 1-/-内皮细胞的内在“血管生成潜力”，uPAR作为“抗血管生成”HKa受体的作用，以及氧化应激对HKa体内抗内皮细胞作用的重要性。我们在mKng 1-/-小鼠中的观察建立了HK作为少数经遗传学证实的血管生成内源性调节因子之一，并且拟议的研究应该为其活性的机制提供重要的见解。公共卫生关系：血管生成在多种临床疾病的发病机制中起着核心作用，特别是癌症和心血管疾病，这是美国两种最常见的死亡原因。例如，血管生成在癌症的发展、进展和转移性扩散中至关重要。在心血管疾病中，血管生成可能具有积极和消极的影响。虽然血管生成对于维持血流和氧气输送到由于潜在的动脉粥样硬化血管疾病而具有受损血流的组织是必不可少的，但血管生成也可能有助于动脉粥样硬化的发展。血管生成也有助于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，参与许多生物学过程，特别是那些炎症起着重要作用的过程。我们已经证明，切割形式的高分子量激肽原（HKa）是一种有效的血管生成抑制剂。我们已经开发出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。在本申请中，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。这些研究应该提供关于这些途径的新信息，这些途径也可能与其他天然存在的血管生成抑制剂的机制相关。