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.