# Redox Regulation of Cu Importer CTR1 in Angiogenesis

> **NIH NIH R01** · AUGUSTA UNIVERSITY · 2023 · $734,105

## Abstract

PROJECT SUMMARY
The aim of this grant is to elucidate the thiol redox modification of the Cu importer, CTR1 as a vital link
between Cu transporters, reactive oxygen species (ROS)-dependent VEGFR2 signaling and reparative
angiogenesis. ROS derived from NADPH oxidase (NOX) act as signaling molecules to promote VEGF-induced
angiogenesis in endothelial cells (ECs) and reparative neovascularization. The fundamental question remains,
how diffusible ROS can activate specific redox signaling to enhance therapeutic angiogenesis? The signaling
function of ROS acts through oxidation of reactive Cys residues in proteins to generate “Cysteine sulfenic acid
(Cys-OH)” (sulfenylation), which is involved in disulfide bond formation and redox signaling. Copper (Cu), an
essential micronutrient, also plays an important role in angiogenesis via unknown mechanisms. The major Cu
entry pathway is via the Cu importer, CTR1, which has only one cytosolic Cys189 in the highly conserved C-
terminal triad, HCH190. The co-investigator of this grant reported that the HCH190 triad acts as a loose “plug” for
Cu entry, and is essential for Cu-induced CTR1 internalization (regulatory endocytosis) which protects against
excess Cu-induced toxicity in HEK cells. However, the mechanistic linkage between CTR1 and VEGF-induced
ROS signaling in mediating angiogenesis in ECs and its in vivo role are entirely unknown. Based on our
preliminary data, we hypothesize that VEGF induces sulfenylation of CTR1 at Cys189 via NOX-derived ROS,
which drives: 1) CTR1 binding to VEGFR2 and their subsequent co-internalization required for activating
sustained VEGFR2 signaling in a Cu transport-independent manner; and 2) Cu entry-dependent
activation of Cu target proteins in ECs. This in turn promotes full angiogenesis and neovascularization
in ischemic diseases. Aim 1 will characterize the VEGF-induced Cys oxidation of CTR1 and determine its role
in angiogenic responses in human and mouse ECs. Aim 2 will determine the mechanisms by which Cys-oxidized
CTR1 activates VEGFR2 signaling and Cu entry-dependent activation of Cu targets in ECs. Aim 3 will determine
the in vivo significance of endothelial CTR1 function in ROS-dependent reparative neovascularization and
address underlying mechanisms using mice hindlimb ischemia and wound healing models. We will use various
innovative reagents, including biotin-labelled Cys-OH trapping probe; BiFC-based protein-protein interaction in
situ and live cell imaging, cell surface biotinylation; and gene transfer of EC-targeted various CTR1 mutants;
newly-developed inducible EC-specific CTR1-/- mice and CRISPR/Cas9-generated CTR1 Cys oxidation-
defective knock-in mutant mice. Highly innovative ICP-Mass Spec, X-ray fluorescence microscopy, Cu
fluorescence probe will be used to analyze intracellular Cu in cells and tissues. Our proposal will provide novel
insights into Cys oxidized CTR1 as a potential therapeutic target for ischemic cardiovascular diseases.

## Key facts

- **NIH application ID:** 10534180
- **Project number:** 5R01HL147550-04
- **Recipient organization:** AUGUSTA UNIVERSITY
- **Principal Investigator:** TOHRU FUKAI
- **Activity code:** R01 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $734,105
- **Award type:** 5
- **Project period:** 2019-12-15 → 2024-11-30

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10534180

## Citation

> US National Institutes of Health, RePORTER application 10534180, Redox Regulation of Cu Importer CTR1 in Angiogenesis (5R01HL147550-04). Retrieved via AI Analytics 2026-06-14 from https://api.ai-analytics.org/grant/nih/10534180. Licensed CC0.

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