Regulation of Angiogenesis by Kininogen

激肽原对血管生成的调节

• 批准号: 8389601
• 负责人: Keith R. McCrae
• 金额: $ 37.34万
• 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 2014-11-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8389601
• 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; Stem cells; Stimulus; Subfamily lentivirinae; Techniques; Three-dimensional analysis; Tissues; United States; Vascular Diseases; Vascular Endothelial Growth Factors; Wound Healing; abstracting; angiogenesis; density; extracellular; improved; in vivo; inhibitor/antagonist; insight; member; mortality; mutant; neovasculature; novel strategies; polypeptide; receptor; reproductive; response; screening; tissue repair; tumor; tumor growth

Abstract 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 antiangiogenic 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.

摘要 在过去的十年里，出现了一个重要的范例，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因子发挥作用。产生这些的亲本蛋白质 多肽通常是凝血和纤溶蛋白家族的成员，或者是 细胞外基质。我们实验室最近的研究集中在裂解高密度脂蛋白的机制上 分子量激肽原(Hka)是内源性凝血途径的一员，可诱导血管内皮细胞的凋亡。 促进内皮细胞增殖，抑制血管生成。尽管Hka抑制血管生成，但最近的研究表明 在BN-KA大鼠中，激肽原基因点突变导致激肽原分泌不足，提示 激肽原缺乏会导致血管生成减少和肿瘤生长。这被归因于 单链高分子激肽原(HK)释放缓激肽(BK)不足，导致 间质BKB2受体活性降低，继而间质血管内皮细胞生长因子分泌减少。至 为了进一步研究这个问题，我们已经缺失了两个小鼠激肽原基因中的一个(MKng1)。筛选 用几种不同的激肽原抗体和敏感的BK免疫印迹mKng1-/-小鼠 放射免疫分析表明，这些动物完全缺乏激肽原。直接地 与BN-KA大鼠不同，mKng1-/-小鼠的初步研究表明，两者的血管生成 肿瘤生长速度加快。我们假设增加mKng1-/-小鼠血管生成的结果 由于在活跃的血管生成部位缺乏生成抗血管生成的Hka。因为，与 大鼠，小鼠的激肽释放酶系统与人类的更接近，我们认为 MKng1-/-小鼠为评估血管生成的调控提供了重要且相关的模型。 激肽原。在这一应用中，我们建议评估潜在的血管生成表型的机制。 MKng1-/-小鼠通过三个特定的目的。在特定的目标1中，我们将比较组织形态、基线 微血管密度、三维血管构筑和血管生成反应 野生型和mKng1-/-小鼠的病理生理刺激。这些研究将使用新开发的， 自动血管计数技术以及分析三维血管的新方法 形态学。在特定的目标2中，我们将确定mKng1-/-小鼠增强的血管生成是否 被补给HK逆转，以及是否需要将HK裂解为Hka才能回归野外- 类型表型。这些研究将使用慢病毒产生的小鼠HK以及抗药性的突变HK 激肽释放酶进行卵裂。在具体目标3中，我们将评估几个重要的潜在机制问题 与mKng1-/-小鼠血管生成前表型的相关性，包括循环内皮细胞水平 祖细胞及其新生血管的能力，mKng1-/-的内在“血管生成潜能” 内皮细胞，uPAR作为“抗血管生成”hka受体的作用，以及氧化的重要性 应激对Hka体内抗内皮细胞作用的影响。我们在mKng1-/-小鼠身上的观察证实香港为 是为数不多的经过基因验证的血管生成内源性调节因子之一，拟议的研究应该 对其活动背后的机制提供了重要的洞察。

项目成果

Lipocalin-2 released in response to cerebral ischaemia mediates reperfusion injury in mice.

lipocalin-2响应脑缺血介导小鼠的再灌注损伤。

• DOI: 10.1111/jcmm.12538
• 发表时间: 2015-07
• 期刊: Journal of cellular and molecular medicine
• 影响因子: 5.3
• 作者: Wang G;Weng YC;Han X;Whaley JD;McCrae KR;Chou WH
• 通讯作者: Chou WH