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Principal Investigator: TOHRU FUKAI
Organization: AUGUSTA UNIVERSITY
Fiscal Year: 2024
Award: $762,300
Funding agency: National Heart Lung and Blood Institute
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.
Terms: <ATAC sequencing><ATAC-seq><ATACseq><Acetylation><Address><Aorta><Apo-E><ApoE><ApoE protein><Apolipoprotein E><Apoptosis><Apoptosis Pathway><Area><Arterial Fatty Streak><Arterial Fatty Streaks><Assay for Transposase-Accessible Chromatin using sequencing><Atheroma><Atheromatous><Atheromatous degeneration><Atheromatous plaque><Atherosclerosis><Atherosclerotic Cardiovascular Disease><Basal Transcription Factor><Basal transcription factor genes><Binding><Bioavailability><Biological Availability><Biosensor><Biotin><Blood Vessels><Blood flow><Body Tissues><CRISPR approach><CRISPR based approach><CRISPR method><CRISPR methodology><CRISPR technique><CRISPR technology><CRISPR tools><CRISPR-CAS-9><CRISPR-based method><CRISPR-based technique><CRISPR-based technology><CRISPR-based tool><CRISPR/CAS approach><CRISPR/Cas method><CRISPR/Cas technology><CRISPR/Cas9><CRISPR/Cas9 technology><Carrier Proteins><Cas nuclease technology><Caveolae><Caveolas><Cell Body><Cell Death><Cell Membrane Lipid Rafts><Cells><Chaperone><Chelating Agents><Chelators><Chemical Fractionation><Citric Acid Cycle><Closure by Ligation><Clustered Regularly Interspaced Short Palindromic Repeats approach><Clustered Regularly Interspaced Short Palindromic Repeats method><Clustered Regularly Interspaced Short Palindromic Repeats methodology><Clustered Regularly Interspaced Short Palindromic Repeats technique><Clustered Regularly Interspaced Short Palindromic Repeats technology><Complexons><Copper><Cu element><Cytosol><Data><Deacetylation><Disease><Disorder><Dysfunction><Endothelial Cells><Endothelium><Enzyme Gene><Enzymes><Exposure to><FRACN><Fluorescence><Fluorescence Light Microscopy><Fluorescence Microscopy><Fractionation><Fractionation Radiotherapy><Functional disorder><Gene Expression><Gene Transfer><General Transcription Factor Gene><General Transcription Factors><Genes><Grant><High Fat Diet><Human><ICP-MS><Inductively Coupled Plasma Mass Spectrometry><Inflammation><Inflammatory><Knock-in><Krebs Cycle><Label><Ligation><Measures><Mediating><Membrane Microdomains><Mice><Mice Mammals><Micronutrients><Mitochondria><Modeling><Modern Man><Molecular><Molecular Chaperones><Molecular Interaction><Morbidity><Morbidity - disease rate><Murine><Mus><Mutant Strains Mice><Mutate><Nuclear><Nuclear Translocation><Oxidation-Reduction><Oxidative Stress><Oxygen Consumption><Phase><Physiologic Availability><Physiopathology><Play><Programmed Cell Death><Proteins><RNA Seq><RNA sequencing><RNAseq><Redox><Roentgen Rays><Role><SIRT1><SIRT1 gene><Short interfering RNA><Sirtuin 1><Site><Small Interfering RNA><Sphingolipid Microdomains><Sphingolipid-Cholesterol Rafts><Staining method><Stains><Synchrotrons><TCA cycle><Therapeutic><Tissues><Transcription Factor Proto-Oncogene><Transcription factor genes><Transfection><Transport Protein Gene><Transport Proteins><Transporter Protein><Tricarboxylic Acid Cycle><Vitamin H><X-Radiation><X-Ray Radiation><X-ray><Xray><assay for transposase accessible chromatin followed by sequencing><assay for transposase accessible chromatin seq><assay for transposase accessible chromatin sequencing><assay for transposase-accessible chromatin with sequencing><atherogenesis><atheromatosis><atherosclerosis plaque><atherosclerotic disease><atherosclerotic lesions><atherosclerotic plaque><atherosclerotic vascular disease><biological sensor><chelation><coenzyme R><endothelial dysfunction><in vivo><innovate><innovation><innovative><insight><knockin><lipid raft><mitochondrial><mitochondrial dysfunction><mortality><mouse mutant><mutant><nano particle><nano-sized particle><nanoparticle><nanosized particle><necrocytosis><novel><oxidation><oxidation reduction reaction><pathophysiology><response><scATAC sequencing><scATAC-seq><scRNA-seq><sensor><shRNA><short hairpin RNA><siRNA><single cell ATAC-seq><single cell ATAC-sequencing><single cell Assay for Transposase Accessible Chromatin sequencing><single cell RNA-seq><single cell RNAseq><single cell expression profiling><single cell sequencing assay for transposase accessible chromatin><single cell transcriptomic profiling><single-cell Assay for Transposase-Accessible Chromatin with sequencing><single-cell RNA sequencing><single-cell assay for transposase-accessible chromatin using sequencing><single-cell assay for transposase-accessible chromatin-seq><small hairpin RNA><social role><therapeutic target><transcription factor><transcriptome sequencing><transcriptomic sequencing><uptake><vascular><vascular inflammation><vulnerable plaque>