Abstract 13464: Antioxidant-1 Plays an Important Role in Wound Repair by Inhibiting Apoptotic Response and Promoting Migration and Proliferation of Endothelial Cells through Akt Phosphorylation in Wound Healing Models
Wound healing involves orchestrated cellular responses, including cell migration, proliferation, and apoptosis, and biological processes, such as inflammation and angiogenesis. Copper (Cu) chelator impairs, and exogenous Cu enhances, wound healing and angiogenesis. However, the mechanisms are not completely understood. We previously demonstrated that Cu transport protein antioxidant-1 (Atox1) is involved in Cu-induced cell growth. Thus, we examined the role of Atox1 in wound healing by using the mouse skin puncture model. In WT mice Atox1 protein and Cu levels are both increased (2 fold and 9 fold at day 7, respectively). Localization of Cu in the wound site was visualized by X-ray fluorescence microscopy and found along the wound edge. Atox1 knockout (Atox1 KO) mice have reduced rate of wound closure (30% closure vs. 80% in WT at day 5) and rescue of Atox1 by lentivirus at the wound site restored wound closure rate. Macrophage number was significantly reduced (mac3+ at day 1) and angiogenesis (CD31+) was strongly impaired in Atox1KO (60% of WT at day 7). Western analysis of tissues from the wounded region shows reduced pAkt levels in Atox1KO that are necessary for anti-apoptotic signaling. Thus we studied the effect of Atox1 knockdown with siRNA on endothelial cell viability. HUVECs treated with siAtox1 showed increased apoptosis by 2-fold (assessed by Annexin V surface expression and DNA fragmentation) in response to pro-inflammatory cytokine TNFα. Phosphorylation of Akt induced by pro-angiogenesis factor VEGF was also reduced without affecting p-ERK or p-p38MAPK. Activation of Akt requires upstream kinase, PI3Kbeta, whose expression was reduced in Atox1 siRNA treated HUVECs. Since Akt is also involved in endothelial migration and proliferation, which is important for wound repair, we examined the role of Atox1 in these responses. Depletion of Atox1 impaired migration towards VEGF (40% of scrambled) and proliferation capacity. In summary, our study demonstrates a novel role of Atox1 as an anti-apoptotic protein of endothelial cells by regulating PI3Kbeta expression and its consequent phosphorylation of Akt, which may contribute to cell migration and proliferation, thereby stimulating wound repair process in response to injury in vivo.
- © 2011 by American Heart Association, Inc.