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.

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.

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National Heart, Lung, and Blood Institute (NHLBI)
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Hemostasis and Thrombosis Study Section (HT)
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Link, Rebecca P
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Cleveland Clinic Lerner
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