Abstract 13384: Copper Transporter ATP7A is Involved in Post-Ischemic Neovascularization by Regulating Endothelial Junctional Integrity Through Binding to IQGAP1
Endothelial junctional integrity is important for neovascularization by regulating cell migration, proliferation, survival, and inflammatory cell infiltration. Copper (Cu), an essential micronutrient, plays an important role in regulating angiogenesis by unknown mechanisms. Cu transporters, including ATP7A, tightly control intracellular levels of Cu. We investigated the role of Cu transporter ATP7A on neovascularization and inflammation, using the mouse hind-limb ischemia model. ATP7A mutant (ATP7Amut) mice have blunted blood flow recovery (60% of WT day 7 blood flow), but augmented macrophage (mac3+) infiltration into ischemic muscle (>2 fold increase vs. WT) 3 days after femoral artery ligation. Increased macrophage infiltration was not associated with adhesion molecule expression, such as ICAM1, VCAM1, and MCP1, or circulating monocyte number. Since endothelial permeability increased by proinflammatory cytokines such as TNFα regulates the amount of monocytes localized in the ischemic region, we hypothesized that ATP7A may be involved in regulating endothelial junctional integrity. Using confluent monolayer of human umbilical vein endothelial cells (HUVECs), we found that knockdown of ATP7A, but not other Cu transport proteins (CTR1 and Atox1), increased gap number and size as visualized by VE-cadherin and beta-catenin immunofluorescence staining basally and following TNF-α stimulation without affecting their protein expression. Further, ATP7A siRNA increased endothelial permeability in both basal state (132% of scrambled siRNA permeability) and following TNFα (124%), as measured by transendothelial electric resistance. Mechanistically, ATP7A binds to IQGAP1, an actin binding scaffold protein that also binds to VE-cadherin/b-catenin and stabilize endothelial junction. Depletion of ATP7A resulted in a mislocalization of IQGAP1, disassembly of actin, as well as increased levels of Rac1 and LIM kinase, regulators of actin cytoskeleton. In summary, ATP7A functions as a novel mediator for inflammatory angiogenesis by regulating endothelial junctional integrity by stabilizing actin via binding to IQGAP1 and modulating regulators of actin cytoskeleton.
- © 2011 by American Heart Association, Inc.