Abstract 18029: Copper Transporter ATP7A Protects Against Endothelial Barrier Dysfunction in Ischemic Injury in Diabetes

Abstract

Endothelial barrier dysfunction is the major vascular complications in inflammatory diseases such as diabetes mellitus (DM). Copper (Cu), an essential micronutrient, plays an important role in physiological repair processes. Since excess Cu has been implicated in various pathophysiologies such as DM, intracellular Cu levels are tightly controlled by Cu exporter ATP7A. In this study, we investigated a role of ATP7A in DM-induced endothelial barrier dysfunction using ischemic limb injury model. Here we show that ATP7A protein expression is markedly upregulated (>2 folds) in ischemic injured tissue, which is significantly decreased in streptozotocin (STZ)-induced type1 DM mice. ATP7A dysfunctional mutant mice or DM mice show increased vascular permeability (assessed by Miles assay), excess macrophage infiltration, and tissue damage in ischemic tissue vs. control mice. In endothelial cells (ECs), ATP7A knockdown with siRNA or ECs derived from DM mice increases EC permeability (132%) as measured by transendothelial electric resistance in a Cu-dependent manner. This is due to decreased protein expression of VE-Cadherin (VE-Cad; 40% decrease), but not beta-catenin, thereby disrupting adherence junctions as visualized by beta-catenin staining. Mechanistically, ATP7A siRNA induces microRNA (miR)-125b (1.8 fold), which reduces VE-Cad protein, in a Cu-dependent manner. Of note, anti-miR125b rescues siATP7A-induced decrease in VE-Cad protein as well as increased EC permeability. ECs or ischemic tissues from DM mice show decreased ATP7A or VE-Cad protein expression and increased miR125b expression (P<0.05). Moreover, ATP7A-depleted ECs exhibit disassembly of actin as visualized by phalloidin through activating Rac1 (1.4 fold), which is inhibited by Cu chelator TTM. Functionally, DM-induced vascular leakage and tissue damage in ischemic limbs is rescued in ATP7A overexpressing transgenic mice treated with STZ. In summary, Cu exporter ATP7A protects against endothelial barrier dysfunction in DM by suppressing miR-125b that downregulates VE-Cad as well as by inhibiting Rac1-mediated actin disassembly. Thus, ATP7A is a potential therapeutic target for promoting tissue repair in diabetic inflammatory ischemic disease.