Copper (Cu), an essential micronutrient, plays a fundamental role in inflammation and angiogenesis; however, its precise mechanism remains undefined. Here we uncover a novel role of Cu transport protein Antioxidant-1 (Atox1), which is originally appreciated as a Cu chaperone and recently discovered as a Cu-dependent transcription factor, in inflammatory neovascularization. Atox1 expression is upregulated in patients and mice with critical limb ischemia. Atox1-deficient mice show impaired limb perfusion recovery with reduced arteriogenesis, angiogenesis, and recruitment of inflammatory cells. In vivo intravital microscopy, bone marrow reconstitution, and Atox1 gene transfer in Atox1−/− mice show that Atox1 in endothelial cells (ECs) is essential for neovascularization and recruitment of inflammatory cells which release VEGF and TNFα. Mechanistically, Atox1-depleted ECs demonstrate that Cu chaperone function of Atox1 mediated through Cu transporter ATP7A is required for VEGF-induced angiogenesis via activation of Cu enzyme lysyl oxidase. Moreover, Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox, thereby increasing ROS-NFκB-VCAM-1/ICAM-1 expression and monocyte adhesion in ECs inflamed with TNFα in an ATP7A-independent manner. These findings demonstrate a novel linkage between Atox1 and NADPH oxidase involved in inflammatory neovascularization and suggest Atox1 as a potential therapeutic target for treatment of ischemic disease.

Purpose: To investigate the role of neutrophil extracellular traps (NETs) and NET-associated proteins in the pathogenesis of oGVHD and whether dismantling of NETs with heparin reduces those changes. Methods: Ocular surface washings from oGVHD patients and healthy subjects were analyzed. Isolated peripheral blood human neutrophils were stimulated to generate NETs and heparinized NETs. We performed in vitro experiments using cell lines (corneal epithelial, conjunctival fibroblast, meibomian gland (MG) epithelial and T cells), and in vivo experiments using murine models, and compared the effects of NETs, heparinized NETs, NET-associated proteins and neutralizing antibodies to NET-associated proteins. Results: Neutrophils, exfoliated epithelial cells, NETs and NET-associated proteins (extracellular DNA, Neutrophil Elastase, Myeloperoxidase, Oncostatin M (OSM), Neutrophil gelatinaseassociated lipocalin (NGAL) and LIGHT/TNFSF14) are present in ocular surface washings (OSW) and mucocellular aggregates (MCA). Eyes with high number of neutrophils in OSW have more severe signs and symptoms of oGVHD. NETs (and OSM) cause epitheliopathy in murine corneas. NETs (and LIGHT/TNFSF14) increase proliferation of T cells. NETs (and NGAL) inhibit proliferation and differentiation of MG epithelial cells. NETs enhance proliferation and myofibroblast transformation of conjunctival fibroblasts. Sub-anticoagulant dose Heparin (100 IU/mL) dismantles NETs and reduces epithelial, fibroblast, T cell and MG cell changes induced by NETs.

Copper (Cu), an essential nutrient, promotes wound healing, however, target of Cu action and underlying mechanisms remain elusive. Cu chaperone Antioxidant-1 (Atox1) in the cytosol supplies Cu to the secretory enzymes such as lysyl oxidase (LOX), while Atox1 in the nucleus functions as a Cu-dependent transcription factor. Using mouse cutaneous wound healing model, here we show that Cu content (by X-ray Fluorescence Microscopy) and nuclear Atox1 are increased after wounding, and that wound healing with and without Cu treatment is impaired in Atox1−/− mice. Endothelial cell (EC)-specific Atox1−/− mice and gene transfer of nuclear-target Atox1 in Atox1−/− mice reveal that Atox1 in ECs as well as transcription factor function of Atox1 are required for wound healing. Mechanistically, Atox1−/− mice show reduced Atox1 target proteins such as p47phox NADPH oxidase and cyclin D1 as well as extracellular matrix Cu enzyme LOX activity in wound tissues. This in turn results in reducing O2− production in ECs, NFkB activity, cell proliferation and collagen formation, thereby inhibiting angiogenesis, macrophage recruitment and extracellular matrix maturation. Our findings suggest that Cu-dependent transcription factor/Cu chaperone Atox1 in ECs plays an important role to sense Cu to accelerate wound angiogenesis and healing.