Abstract 13138: Antioxidant-1, a Copper Transport Protein, promotes Inflammatory Neovascularization via Chaperone and Transcription Factor Function

Abstract

Copper (Cu), an essential micronutrient, has been implicated in inflammation and angiogenesis with unknown mechanism. Cu transport protein, antioxidant-1 (Atox1) functions not only as Cu chaperone to activate secretory Cu enzymes (including lysyl oxidase (LOX)) via Cu transporter ATP7A, but also as Cu-dependent transcription factor. However, role of Atox1 in inflammatory neovascularization has not been investigated. Here we show that Atox1 expression is markedly upregulated in patients and mice with critical limb ischemia. Together with immunohistochemistry, micro-CT analysis and sponge implant assay, Atox1 KO mice exhibit impaired ischemia induced perfusion recovery, arteriogenesis/angiogenesis and inflammatory cell infiltration (46%). Furthermore, bone marrow (BM) reconstitution and adenovirus mediated Atox1 gene transfer in Atox1 KO mice show that Atox1 in tissue resident endothelial cells (ECs), but not in BM cells, is essential for neovascularization and recruitment of inflammatory cells which release angiogenic cytokines TNFα and VEGF. Mechanistically, Atox1 depletion of mice or ECs shows inhibition of ischemia (3.4-fold) and VEGF (1.6 fold) induced extracellular matrix/secretory Cu enzyme LOX activity in Cu/ATP7A-dependent manner, resulting in impaired (42%) capillary tube formation. Moreover, Atox1 knockdown in ECs inhibits ROS production (2.5 fold) and its downstream NFκB-DNA binding, VCAM1/ICAM1 expression and monocyte adhesion in ECs activated by TNFα, in Cu-dependent and ATP7A-independent manner, which are further supported by in vivo DHE staining and Bioluminescence imaging of NFκB reporter mice. Of note, Atox1 translocates to the nucleus (90%) in response to TNFα, thereby increasing transcription of p47phox NADPH oxidase by binding to its promoter in Cu-dependent manner. p47phox re-expression rescued reduced TNFα -induced VCAM1 expression in Atox1 KO ECs. In summary, Atox1 plays an important role in inflammatory neovascularization via Cu chaperone for LOX to increase angiogenesis as well as transcription factor for p47phox to promote ROS and inflammatory responses. Thus, Atox1 is a potential therapeutic target for promoting tissue repair in ischemic cardiovascular disease.