Sunday, May 18, 2025
5/18/2025
Cu uptake transporter as a disturbed flow sensor in vascular inflammatory disease - PROJECT SUMMARY The aim of this grant is to elucidate the novel role of endothelial Cu uptake transporter CTR1 as a “mechanosensor” that promotes Cu-dependent inflammation and cuproptosis involved in atherosclerosis. Oxidative stress, inflammation, and mitochondrial (mito) dysfunction in endothelial cells (ECs) contributes to atherosclerosis, predominantly occurring in arterial regions exposed to disturbed blood flow (d- flow), while those in the stable laminar flow (s-flow) regions are protected. The mechanisms by which d-flow and s-flow regulate atherogenesis are still poorly understood. Copper (Cu), an essential micronutrient, is greatly increased in human atherosclerotic plaques, while Cu promotes, and Cu chelation inhibits atherosclerosis in mice via unknown mechanisms. The bioavailability of intracellular Cu is tightly controlled by Cu transport and Cu chaperone proteins including Cu uptake transporter CTR1 and cytosolic Cu chaperone Atox1 that also functions as a Cu-dependent transcription factor to upregulate inflammatory gene expression. Recent evidence reveals that excess Cu induces a new type of programmed cell death, termed “cuproptosis”, characterized by decrease in lipoylated TCA cycle proteins and Fe-S cluster proteins, resulting in mito dysfunction. However, the roles of Cu and endothelial CTR1 in d-flow-induced mechano-signaling, inflammation, mito dysfunction and any involvement of cuproptosis are entirely unknown. Our preliminary data are consistent with the hypothesis that endothelial CTR1 funcions as a novel disturbed flow “mechanosensor” that orchestrates cytosolic Cu- mediated Atox1 nuclear translocation via ROS-dependent acetylation leading to inflammation (early phase) as well as mito Cu accumulation following CTR1 binding to mitoCu transporter, leading to cuproptosis (late phase), which contributes to atherosclerosis. Aim 1 will establish the role of CTR1 as a d-flow sensor to drive Cu- and ROS-dependent Atox1 nuclear translocation and inflammation and address molecular mechanisms in cultured ECs. Aim 2 will determine whether d-flow induces mito dysfunction and cuproptosis via increasing mitoCu following CTR1 binding to mitoCu transporter SLC25A3 in ECs. Aim 3 will determine the role of endothelial CTR1 in vascular inflammation, cuproptosis and flow-dependent atherosclerosis and address underlying mechanisms in vivo. We will use inducible EC-specific Ctr1-/- or -Atox1-/- or -SLC25a3-/- mice or newly developed CRISPR/Cas9-generated “acetylation dead” Atox1 knock-in mutant mice with high fat diet or partial carotid ligation to induce atherosclerosis. We will also use compartment-specific redox-sensitive biosensors; biotin-labeled CysOH trapping probe; scRNAseq and scATACseq; newly developed, highly specific mito-targeted Cu-depleting nanoparticle (mitoCDN); highly innovative ICP-Mass Spec, X-ray fluorescence microscopy and mito-targeted Cu fluorescence probes to measure Cu levels in cells or tissues. Our proposal will provide new insights into endothelial CTR1 as a potential therapeutic target for treatment of flow- and Cu- dependent atherosclerosis.
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