Role of Cu Transporter in EC Metabolism, ROS, and Atherosclerosis

Project Summary The goal of Project 1 is to demonstrate that disturbed endothelial copper (Cu) metabolism drives endothelial dysfunction and atherosclerosis by integrating excess glycolysis and mitochondrial (mito)ROS-epigenetic crosstalk. Cardiovascular risk factors such as inflammation, oxidative stress, and diabetes contribute to endothelial dysfunction and atherosclerosis. By contrast, aerobic glycolysis regulated by glycolytic enzymes including PFKFB3 is required for normal endothelial cell (EC) function and its dysregulation contributes to endothelial dysfunction. However, the key mechanisms linking EC metabolism, ROS, and atherosclerosis remain unclear. Cu, an essential micronutrient, is highly increased in human atherosclerosis, but its role is controversial, based on Cu chelation and optimal dietary Cu intake studies. Since excess Cu is toxic, Cu bioavailability is tightly controlled by Cu transporting ATPase (ATP7A). However, the role of endothelial ATP7A and its molecular linkage with Cu metabolism, oxidative stress, and glycolysis involved in atherosclerosis are entirely unknown. Preliminary data show that ATP7A expression was dramatically decreased in mouse atherosclerotic aorta or human ECs exposed to pro-atherogenic disturbed flow. Unexpectedly, ATP7A knockdown in human ECs with bulk RNAseq indicated induction of the endothelial-to-mesenchymal transition (EndMT) which promotes EC dysfunction and atherosclerosis. Mechanistically, loss of ATP7A in ECs increased [Cu]i, which in turn promoted excess PFKFB3/glycolysis-ROS axis as well as mitoROS-mediated upregulation of histone demethylase JMJD2B-TGF2-Snail axis to induce EndMT. In vivo, inducible EC-specific ATP7A-/-(iEC-ATP7A-/-)/ApoE-/- mice showed induction of EndMT and enhanced atherosclerosis. Our central hypothesis is that endothelial ATP7A dysfunction drives EndMT by orchestrating the PFKFB3-glycolysis-ROS axis as well as epigenetic reprogramming via mitoROS-JMJD2B axis in a Cu-dependent manner, leading to Snail induction, which in turn accelerates atherosclerosis. Aim 1 will determine whether endothelial ATP7A dysfunction drives EndMT in a Cu-, glycolysis- and ROS-dependent manner in cultured ECs and address underlying mechanisms. Aim 2 will determine whether endothelial ATP7A dysfunction accelerates EndMT and atherosclerosis in a Cu-, glycolysis- and ROS-dependent manner in vivo. We will use newly developed iEC-ATP7A-/-/EC-tracing mTmG mice, highly innovative CRISPR/Cas9-generated knock-in mutant mice that reduce glycolysis in the endothelium (iEC-Glyco-Lo), iEC-Cu importer Ctr1-/- or Pfkfb3-/- mice, carotid partial ligation to induce atherosclerosis, compartment-specific redox-sensitive biosensors, and transcriptomics and metabolomics. Highly innovative ICP- Mass Spec and X-ray fluorescence microscopy will be used to analyze Cu metabolism in cells and tissues. Our proposal will provide new insights into targeting excess Cu-glycolysis-ROS (mitoROS) axis or enhancing endothelial ATP7A function as new therapeutic approaches for treatment of various cardiovascular diseases. Project 1 is highly dependent on the expertise and support provided by other Projects and Cores of this PPG.