Abstract
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.
Highlights
Copper (Cu), an essential micronutrient and catalytic cofactor, plays an important role in physiological repair processes including wound healing and angiogenesis as well as various pathophysiologies including tumor growth and inflammatory diseases such as atherosclerosis[1,2,3,4,5]
Irradiated Atox1−/− mice transplanted with WT-Bone marrow (BM), but not irradiated WT mice reconstituted with Atox1−/− BM, showed impaired limb perfusion recovery compared to control group (WT mice reconstituted with WT-BM) (Fig. 1C)
To determine the mechanism by which Atox[1] is involved in reactive oxygen species (ROS) production in endothelial cells (ECs), we examined a role of Atox[1] in expression of NADPH oxidase, a major source of ROS involved in angiogenesis[31]
Summary
Copper (Cu), an essential micronutrient and catalytic cofactor, plays an important role in physiological repair processes including wound healing and angiogenesis as well as various pathophysiologies including tumor growth and inflammatory diseases such as atherosclerosis[1,2,3,4,5]. We demonstrate that Atox[1] plays an essential role in inflammatory neovascularization via Cu chaperone function to increase angiogenesis via regulating extracellular matrix (ECM) modifying secretory Cu enzyme lysyl oxidase (LOX) activity as well as transcription factor function to promote ROS production and inflammatory responses via increasing NADPH oxidase organizer p47phox expression. We used Atox1−/− mice with hindlimb ischemia model and sponge implant model, and various approaches including micro-CT, in vivo intravital microscopy, gene transfer, bone marrow transplantation (BMT), in situ O2− detection, bioluminescence imaging of NFκ B reporter mice, and in vitro reporter gene assay, and chromatin immunoprecipitation (ChIP) assay These findings demonstrate a novel linkage between Cu transport protein Atox[1] and NADPH oxidase involved in inflammatory angiogenesis and provide insight into Atox[1] as a potential therapeutic target for inflammation-dependent ischemic cardiovascular diseases
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