Abstract Body (Do not enter title and authors here): Background: Neovascularization in response to ischemia relies on reactive oxygen species (ROS), inflammatory, and angiogenic signaling in endothelial cells (ECs). Our prior study suggested that the copper (Cu) chaperone Atox1 acts as a Cu-dependent transcription factor for p47phox, facilitating ROS-NFkB-dependent inflammatory responses in ECs stimulated by inflammatory cytokine TNFα. However, the precise mechanism of Atox1’s nuclear translocation during inflammatory angiogenesis is unknown. Posttranslational SUMOylation and deSUMOylation by SENP1 in the cytosol and nucleus play crucial roles in regulating gene expression, protein subcellular localization and signal transduction. In silico analysis identified a conserved potential SUMOylation motif at Lys(K3) of Atox1.

Results: Here we show that TNFα or hypoxia stimulation in human aortic ECs, as well as mouse hindlimb ischemia model that promotes inflammatory angiogenesis, significantly increased Atox1-K3 SUMOylation. Mechanistically, ROS induced by TNFα or hypoxia led to the oxidation/inactivation of SENP1 at Cys603 in the cytosol, thereby elevating Atox1 K3-SUMOylation (3.6-fold). Unexpectedly, Atox1siRNA or overexpression of “SUMO-dead” Atox1K3R mutant in ECs inhibited TNFα or hypoxia-induced, activation of NFkB (increased cytosolic p-NFkB and its nuclear translocation) as well as Cu-dependent Atox1 nuclear translocation. Additionally, in the nucleus under reducing conditions, ChIP and promoter analysis revealed that deSUMOylation of Atox1 by nuclear active SENP1 is required for Atox1’s transcriptional activity on inflammatory and angiogenic genes VCAM/ICAM, IL-8 and RANTES. Functionally, siAtox1 and “SUMO-dead” Atox1K3R mutant reduced inflammatory and angiogenic responses, including monocyte adhesion, migration and capillary tube formation. In vivo, EC-specific Atox1^-/-mice or CRISPR/Cas9-generated “SUMO-dead” Atox1K3R knock-in mutant mice exhibited significantly reduced blood flow recovery, Mac3+ inflammatory cells, and angiogenesis (CD31+ capillary) in hindlimb ischemia and sponge implant inflammatory angiogenesis models.

Conclusions: SUMOylation of Atox1 via oxidative inactivation of SENP1 in the cytosol promotes p-NFkB and the nuclear translocation of both Atox1 and NFkB. Within the nucleus, deSUMOylated Atox1 and NFkB synergistically enhance the expression of inflammatory and angiogenic genes in inflamed or hypoxic ECs, thereby driving neovascularization in ischemic cardiovascular diseases.