Endothelial Cu Exporter Atp7a Deficiency Promotes Cu-Dependent Endothelial-to-Mesenchymal Transition and Atherosclerosis via Orchestrating Glycolysis and Ros Signaling

Introduction: Oxidative stress, inflammation and metabolic reprogramming contribute to endothelial cell (EC) dysfunction and atherosclerosis. Copper (Cu), an essential micro-nutrient, promotes atherosclerosis via unknown mechanism. Since excess Cu is toxic, bioavailability of intracellular Cu is tightly controlled by Cu exporter ATP7A. However, the role of ATP7A and its molecular linkage with inflammation, oxidative stress and metabolism in EC dysfunction and atherosclerosis are entirely unknown. Results: Here we found a dramatic reduction of ATP7A expression in human and mouse atherosclerotic aorta. Surprisingly, ATP7A knockdown (KD) in human aortic ECs using shRNA changed the cell morphology from EC to mesenchymal types. This was confirmed by RNAseq/qPCR showing reduced EC markers and increased mesenchymal markers, indicating endothelial to mesenchymal transition (EndMT). Mechanistically, loss of ATP7A in ECs increased [Cu]i and PFKFB3-dependent glycolysis (1.8- fold by ECAR in Seahorse assay). Overexpression of PFKFB3 or constitutively active PFK2, which enhances glycolysis, or direct Cu treatment induced EndMT. In parallel, ATP7A KD in ECs increased mitochondrial (mito)ROS (9.8 fold)-histone demethylase JMJD2B, which induced inflammatory EndMT inducer TGFβ2-EndMT transcription factor Snail (2 fold) axis. Of note, ATP7A KD-induced glycolysis as well as mitoROS- TGFβ2-Snail axis involved in EndMT were inhibited by Cu chelator TTM. In vivo, Cu transport dysfunctional ATP7A mutant/ApoE -/- mice showed enhanced atherosclerosis and CD31 + /αSMA + cells representing EndMT in aorta compared to control ApoE -/- mice, which was further confirmed by EC-specific ATP7A -/- /mTmG reporter mice. Conclusion: Our study demonstrates that ATP7A dysfunction in ECs drives EndMT by orchestrating mitoROS-JMJD2B-TGFβ as well as PFKFB3-glycolysis axis in a Cu dependent manner, leading to Snail induction to accelerates atherosclerosis. Thus, ATP7A is a potential therapeutic target for treatment of inflammatory vascular disease.