Human cells transport dehydroascorbic acid through facilitative glucose transporters, in apparent contradiction with evidence indicating that vitamin C is present in human blood only as ascorbic acid. On the other hand, activated host defense cells undergoing the oxidative burst show increased vitamin C accumulation. We analyzed the role of the oxidative burst and the glucose transporters on vitamin C recycling in an in vitro system consisting of activated host-defense cells co-cultured with human cell lines and primary cells. We asked whether human cells can acquire vitamin C by a "bystander effect" by taking up dehydroascorbic acid generated from extracellular ascorbic acid by neighboring cells undergoing the oxidative burst. As activated cells, we used HL-60 neutrophils and normal human neutrophils activated with phorbol 12 myristate 13-acetate. As bystander cells, we used immortalized cell lines and primary cultures of human epithelial and endothelial cells. Activated cells produced superoxide anions that oxidized extracellular ascorbic acid to dehydroascorbic acid. At the same time, there was a marked increase in vitamin C uptake by the bystander cells that was blocked by superoxide dismutase but not by catalase and was inhibited by the glucose transporter inhibitor cytochalasin B. Only ascorbic acid was accumulated intracellularly by the bystander cells. Glucose partially blocked vitamin C uptake by the bystander cells, although it increased superoxide production by the activated cells. We conclude that the local production of superoxide anions by activated cells causes the oxidation of extracellular ascorbic acid to dehydroascorbic acid, which is then transported by neighboring cells through the glucose transporters and immediately reduced to ascorbic acid intracellularly. In addition to causing increased intracellular concentrations of ascorbic acid with likely associated enhanced antioxidant defense mechanisms, the bystander effect may allow the recycling of vitamin C in vivo, which may contribute to the low daily requirements of the vitamin in humans.

Key Words: ADAMs Ⅲ metalloproteinase-disintegrins Ⅲ TNF␣-convertase Ⅲ proliferative retinopathy Ⅲ pathological neovascularization P athological neovascularization has a critical role in diseases such as cancer, 1,2 rheumatoid arthritis 3 and proliferative retinopathies, including retinopathy of prematurity, diabetic retinopathy and the wet form of macular degeneration. 4,5 Therefore molecules with roles in pathological neovascularization are considered potential targets for treatment of these conditions. Previous studies have identified a role for the cell surface metalloproteinase ADAM17 (a disintegrin and metalloproteinase 17, also referred to as TACE [tumor necrosis factor ␣-converting enzyme]) in crosstalk between the VEGFR2 and extracellular signal-regulated kinase 1/2 in endothelial cells, and in processing several receptors with key functions in angiogenesis, including the VEGFR2 and Tie2. 6 The goal of the present study was to determine whether ADAM17 has a role in angiogenesis or pathological neovascularization in vivo by subjecting conditional knockout mice carrying floxed alleles of ADAM17 7 and a Cre-recombinase expressed either in endothelial cells (Tie2-Cre) or in smooth muscle cells and pericytes (␣-smooth muscle actin [␣sma] Cre) to mouse models of pathological neovascularization.ADAM17 was first discovered as the converting enzyme for tumor necrosis factor (TNF)␣, 8,9 a potent proinflammatory cytokine that is a causative factor in autoimmune diseases such as rheumatoid arthritis and Crohn's disease as well as in septic shock in mice. 10 Once mice lacking ADAM17 were generated, it became clear that ADAM17 is Original received August 14, 2009; revision received January 13, 2010; accepted January 19, 2010 The ability of ADAM17 to release endothelial cell membrane proteins on stimulation with VEGF-A raised questions about what role ADAM17 has during developmental angiogenesis and in pathological neovascularization in adult animals. Although mice lacking ADAM17 die perinatally, most likely as a consequence of their severe heart valve defects, 11,12 there have been no reports of defects in developmental angiogenesis in these animals. To address whether ADAM17 has a role in angiogenesis or pathological neovascularization or both, we conditionally inactivated ADAM17 in endothelial cells or in smooth muscle cells such as pericytes, and then determined how lack of ADAM17 affects two mouse models for pathological neovascularization, the oxygen induced retinopathy model for retinopathy of prematurity, and growth of heterotopically injected tumor cells. Moreover, we assessed proliferation and chord formation of endothelial cells lacking ADAM17, and evaluated the role of ADAM17 in the proteolytic release of membrane proteins with known roles in angiogenesis and pathological neovascularization. Methods Reagents and Cell LinesPorcine aortic endothelial cells expressing VEGFR2/KDR (PAE/ KDR cells) and mouse embryonic fibroblasts (mEFs) lacking ADAM17 have been described previously. 6,15 Reagents were from...

Lymph node expansion during immune responses is accompanied by rapid vascular expansion. The reestablishment of quiescence and stabilization of the newly expanded vasculature and the regulatory mechanisms involved have not been well studied. We show that while initiation of vascular expansion in immune-stimulated nodes is associated with upregulated endothelial cell proliferation, increased high endothelial venule trafficking efficiency and VCAM-1 expression, and disrupted perivascular fibroblastic reticular cell organization, the reestablishment of vascular quiescence and stabilization after expansion is characterized by reversal of these phenomena. While CD11cmed cells are associated with the initiation of vascular expansion, CD11chiMHCIImed dendritic cells accumulate later and their short-term depletion in mice abrogates the reestablishment of vascular quiescence and stabilization. CD11chiMHCIImed cells promote endothelial cell quiescence in vitro and, in vivo, mediate quiescence at least in part by mediating reduced lymph node VEGF. Disrupted vascular quiescence and stabilization in expanded nodes is associated with attenuated T cell-dependent B cell responses. These results describe a novel mechanism whereby CD11chiMHCIImed dendritic cells regulate the reestablishment of vascular quiescence and stabilization after lymph node vascular expansion and suggest that these dendritic cells function in part to orchestrate the microenvironmental alterations required for successful immunity.