Regulation of Angiogenesis by Kininogen

描述(申请人提供)：在过去的十年里，一个重要的范例已经出现，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物发挥作用。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，或者是细胞外基质的成分。本实验室最近的研究主要集中在内源性凝血途径中的高分子激肽原(Hka)裂解诱导增殖的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，但在BN-KA大鼠中，最近的研究表明，激肽原基因的点突变导致激肽原分泌不足，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)释放缓激肽(BK)不足，导致间质BK B2受体激活减弱，从而导致间质VEGF分泌减少。为了进一步研究这个问题，我们删除了两个小鼠激肽原基因中的一个(MKng1)。用几种不同的激肽原抗体和灵敏的BK放射免疫分析对mKng1-/-小鼠进行免疫印迹筛选，结果表明这些动物完全缺乏激肽原。与BN-KA大鼠直接对比的是，在mKng1-/-小鼠身上的初步研究表明，血管生成和肿瘤生长都增加了。我们假设mKng1-/-小鼠的血管生成增加是由于在活跃的血管生成部位缺乏生成抗血管生成的Hka所致。由于与大鼠相比，小鼠的激肽释放酶系统更接近于人类，我们认为mKng1-/-小鼠为评估激肽原对血管生成的调控提供了一个重要的相关模型。在这个应用中，我们建议通过三个特定的目标来评估mKng1-/-小鼠血管生成前表型的潜在机制。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、三维血管构筑以及血管生成对病理生理刺激的反应。这些研究将使用新开发的自动血管计数技术，以及分析三维血管形态的新方法。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HK的补充逆转，以及HK到Hka的切割是否是恢复到野生型表型所必需的。这些研究将使用慢病毒产生的小鼠HK，以及抵抗激肽释放酶切割的突变HK。在具体目标3中，我们将评估几个可能与mKng1-/-小鼠的前血管生成表型相关的重要机制问题，包括循环内皮祖细胞的水平及其新生血管的归巢能力，mKng1-/-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，以及氧化应激在体内Hka抗内皮细胞效应中的重要性。我们在mKng1-/-小鼠身上的观察证实，香港是少数几个经过基因验证的血管生成内源性调节因子之一，拟议的研究应该为其活动背后的机制提供重要的见解。公共卫生相关性：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，这两种疾病是美国最常见的死亡原因。例如，血管生成在癌症的发展、进展和转移扩散中起着至关重要的作用。在心血管疾病中，血管生成可能有积极和消极的影响。由于潜在的动脉粥样硬化性血管疾病，血管生成对于维持血流和向血流受损的组织输送氧气是必不可少的，但血管生成也可能促进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，参与许多生物过程，特别是那些在炎症中起重要作用的过程。我们已经证明，裂解形式的高分子激肽原(Hka)是一种有效的血管生成抑制因子。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都有所增加。在这一应用中，我们希望扩展这些研究，并确定Hka抑制完整生物体血管生成的机制。这些研究应该提供有关这些途径的新信息，这些信息可能也与其他自然发生的血管生成抑制剂的机制有关。

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.