Abstract 15774: Cu Uptake Transporter CTR1 Cys189 Oxidation Protects Against Atherosclerosis via Limiting Mitochondrial Dysfunction and Endothelial Senescence

Abstract

Background: Copper (Cu) is an essential micronutrient, but excess Cu is toxic. Role of copper in atherosclerosis remains unclear. The major Cu-uptake transporter CTR1 provides Cu to Cu chaperone/transcription factor Atox1 to promote inflammation as well as mitochondrial cytochrome C oxidase (COX) to regulate mitochondrial (mito)function. We reported that growth factor VEGF induced CTR1 Cys189 oxidation promotes VEGFR2/CTR1 internalization and angiogenesis in endothelial cells (ECs) while excess Cu induces CTR1 internalization to prevent Cu toxicity. However, role of CTR1-Cys189 in ROS-dependent mito dysfunction and senescence in ECs involved in atherosclerosis remains unknown. Results: Here we showed that inducible EC-specific Ctr1 -/- /ApoE -/- mice with high fat diet (HFD) reduced atherosclerosis (72%) and Mac3+ inflammatory cell (62%) vs. atherosclerotic ApoE -/- /HFD mice aorta which showed Ctr1-CysOH formation vs. control ApoE -/- mice. CRISPR-generated “redox-dead” Ctr1-Cys189A knock-in ApoE -/- /HFD mice aorta inhibited Ctr1-CysOH formation but enhanced atherosclerotic lesions and Cu accumulation (ICP-MS) vs. ApoE -/- /HFD mice. This in turn enhanced senescence (β-gal and markers), DNA oxidation, macrophage recruitment and mitochondrial hyperfusion (by TEM). In cultured human EC, proatherogenic cytokine TNFα stimulation rapidly induced CTR1-CysOH formation and CTR1 internalization, which were inhibited by CTR1-C189A or Nox4 shRNA. CTR1-C189A overexpression increased intracellular Cu and its downstream p-Drp1-S637 that promotes mitochondrial hyperfusion, mito dysfunction and mitoROS. This was associated with CTR1-C189A binding to mito Cu transporter SLC25A3, which might facilitate mito Cu overload and mito dysfunction. These CTR1-C189A-induced responses were inhibited by Cu chelator TTM and mito O 2 - scavenger Mito-TEMPO. Conclusions: CTR1-Cys189 oxidation-induced internalization limits excess Cu entry to protect against pro-atherogenic phenotype of CTR1/Cu. Preventing CTR1-Cys189 oxidation increases mito dysfunction via enhancing novel Cu-pS637-Drp1 axis as well as CTR1-SLC25A3 axis, which promotes mitoROS and EC senescence, thereby accelerating atherosclerosis.