Ferritin is an iron binding protein that is present in both intracellular and extracellular compartments. Levels of ferritin are markedly elevated in inflammation and malignancy. Our work to understand the binding partners of ferritin led us to identify H-kininogen (HK, high molecular weight kininogen) and its cleavage product, HKa, as ferritin binding proteins. HK is a key protein that modulates hemostasis, inflammation, and vascular remodeling. We have shown that HK/HKa binds to ferritin and that ferritin blocks the apoptotic effects of HKa on endothelial cells. These results suggest that through its effects on HK/HKa, ferritin may promote vascular remodeling and angiogenesis. These findings expand the role of ferritin beyond that of iron storage, and forge a new link between HK/HKa, ferritin, and angiogenesis. The goal of this proposal is to elucidate the mechanism of the interaction between ferritin and HK/HKa, and to understand its biological consequences.
Our Specific Aims are to (1) Identify the mechanism(s) by which ferritin regulates the activity of HK/HKa on endothelial cells. Specifically, we will test the effect of ferritin on adhesive signaling and HKa- induced oxidant stress. (2) Determine how ferritin regulates inhibitory effects of HK/HKa on angiogenesis. Using in vitro models, we will test whether ferritin stimulates migration or organization of HKa-treated endothelial cells into blood vessels. We will also examine whether ferritin reverses the HKa-mediated inhibition of angiogenesis in vivo. (3) Identify the role of specific amino acid domains in mediating the interaction between HK/HKa and ferritin. We will map HK domains required for ferritin binding, evaluate characteristics of ferritin important to regulation of HK activity, and define critical intra- and intermolecular contacts in HK and the HK-ferritin complex using solution state analysis. Collectively, these experiments will define for the first time the role of ferritin in basic biological mechanisms involved in the control of vascular remodeling and angiogenesis, and identify specific targets in both ferritin and HK/HKa that can be used to modulate these processes. Lay summary: Angiogenesis is the formation of new blood vessels from existing blood vessels. This process must be carefully regulated, because excessive blood vessel formation can contribute to diseases such as diabetes and cancer. On the other hand, insufficient angiogenesis can also have negative effects, contributing to poor wound healing. We have found that ferritin, a naturally occurring protein, may play a previously unsuspected role in regulating blood vessel formation. We propose to test how this occurs. Ultimately, this will improve our understanding of blood vessel formation in health and disease, and may lead to ways to control this process. ? ? ?
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