Ivana De Domenicoa,b, Diane McVey Wardb, and Jerry Kaplanb,1 Departments of aInternal Medicine and bPathology, School of Medicine, University of Utah, Salt Lake City, UT 84132-2501

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erritin is a protein whose principal role within cells is the storage of iron in a nontoxic, but bioavailable, form. The assembled ferritin molecule, often referred to as a nanocage, can store up to 4,500 atoms of iron (for review see ref. 1). In contrast to cytosolic ferritin, serum ferritin is relatively iron-poor and may contain only a few atoms of iron. Ferritin does not have an obvious leader sequence, and the mechanism of its release from cells is unclear. Serum ferritin is increased during inflammation, which suggests that it may play a role in modulating inflammatory effects (2, 3). In a recent issue of PNAS, Coffman et al. (4) provide compelling evidence that serum ferritin regulates vascular remodeling and angiogenesis, demonstrating a role for serum ferritin in cell proliferation. Angiogenesis is a physiological process involving the formation of new blood vessels from preexisting vessels. Inflammation can regulate angiogenesis through the cleavage of kininogen (HK), a 120-kDa single-chain plasma glycoprotein (5). Activation of the plasma protease kallikrein leads to cleavage of HK and the production of 2 cleavage products bradykinin (BK) and cleaved HKa. BK is known to be an angiogenesis stimulator, whereas HKa is a potent angiogenesis inhibitor inducing the apoptosis of proliferating endothelial cells (Fig. 1A). Coffman et al. (4) demonstrate that the inhibitory activity of HKa can be blocked by serum ferritin. Addition of HKa to cultured endothelial cells results in a loss of endothelial cell viability in a dose-dependent manner. Serum ferritin prevents HKa-mediated endothelial cell death, permitting the endothelial cells to organize into blood vessels. Ferritin added to endothelial cells culture did not directly affect vascular remodeling, rather ferritin binding to HKa inhibited HKa effects on cells (Fig. 1B). Coffman et al. also demonstrated that ferritin reverses the HKa-mediated inhibition of angiogenesis in an in vivo model of tumor xenografts. Together, the in vitro and in vivo data suggest that the interaction between HKa and ferritin contributes to regulation of angiogenesis, particularly during tumor growth. Coffman et al. (4) identified the site on HKa that binds ferritin. The precursor HK contains 6 functionally different domains. Cleavage of HK by kallikrein rewww.pnas.org兾cgi兾doi兾10.1073兾pnas.0813318106

Fig. 1. Schematic of HK cleavage and the role of serum ferritin in angiogenesis. (A) Cleavage of HK by the plasma protease kallikrein releases the angiogenesis stimulator BK and the angiogenesis inhibitor HKa. (B) Endothelial cells form new blood vessels. High concentrations of HKa stop blood vessel formation by inducing apoptosis of activelyproliferating endothelial cells. Binding of ferritin to HKa inhibits HKa activity and promotes endothelial cell proliferation with consequent blood vessel formation.

sults in the generation of BK (domain 4) and HKa, which is composed of 2 proteins that are disulfide linked that are organized into the heavy chain (domains 1–3) and light chain (domains 5 and 6). Domain 5 of HKa has been reported to inhibit proliferation of endothelial cells through binding to cell surface receptors (6). Coffman et al. identified a 22-aa region in domain 5 of HKa that binds ferritin. This site binds iron-poor ferritin and iron-rich ferritin, with an affinity in the low nanomolar range. Ferritin binding to HKa might preclude HKa binding to cells, therefore allowing cell proliferation and angiogenesis. The ferritin multimer consists of 24 subunits of different amounts of either H-ferritin or L-ferritin monomers. The H-ferritin monomer contains the ferroxi-

dase activity that is required to insert iron into the nanocage. The L-ferritin monomer helps provide stability to the assembled nanocage. The level of serum ferritin is markedly elevated in inflammation, malignancy, and iron overload disorders (7). Indeed, the level of serum ferritin is used clinically to assess iron overload disease and the efficacy of iron chelation or therapeutic phlebotomy. The function of extracellular ferritin, however, has been obscure. Older studies have suggested that serum ferritin can provide iron for cell growth but cell surface receptors for ferritin have not been described in molecular terms. Recently, Li et al. (8) have provided strong evidence that binding of the (relatively) iron-poor serum ferritin to the surface receptor Scara5 provides iron to specific subsets of developing kidney cells. Serum ferritin levels are increased by inflammation, suggesting ferritin may modulate inflammation or immunity (9). H-ferritin chains are transcriptionally activated through cytokines such as TNF-␣ (10). The observation that serum ferritin can bind to TIM-2 receptors on mouse lymphocytes suggests that it might be a signal for inflammation (11). The finding by Coffman et al. (4) that serum ferritin may regulate vascular remodeling and angiogenesis adds further support to that view. It might be informative to determine which ferritin subunit, H or L, is involved in the binding to HKa, because the mouse lymphocyte receptor TIM-2 seems to prefer H-enriched ferritin, whereas Scara5-mediated ferritin uptake prefers L-chain-enriched ferritin. Coffman et al. (4) provide new insight into the modulation of the antiangiogenic mechanism of HKa. Their work reveals how the antiangiogenesis and proangiogenesis effects of kallikrein cleavage products BK and HKa are regulated. What remains to be determined to understand the proangiogenic effect of ferritin is the conditions, cell types, and mechanisms of ferritin release. ACKNOWLEDGMENTS. This work was supported by National Institutes of Health Grants DK070947 and DK30534.

Author contributions: I.D.D., D.M.W., and J.K. wrote the paper. The authors declare no conflict of interest. See companion article on page 570 in issue 2 of volume 106. 1To

whom correspondence should be addressed. E-mail: [email protected].

PNAS 兩 February 10, 2009 兩 vol. 106 兩 no. 6 兩 1683–1684

COMMENTARY

Serum ferritin regulates blood vessel formation: A role beyond iron storage

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