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Endothelial Antioxidant-1: a Key Mediator of Copper-dependent Wound Healing in vivo

View Article: PubMed Central - PubMed

ABSTRACT

Copper (Cu), an essential nutrient, promotes wound healing, however, target of Cu action and underlying mechanisms remain elusive. Cu chaperone Antioxidant-1 (Atox1) in the cytosol supplies Cu to the secretory enzymes such as lysyl oxidase (LOX), while Atox1 in the nucleus functions as a Cu-dependent transcription factor. Using mouse cutaneous wound healing model, here we show that Cu content (by X-ray Fluorescence Microscopy) and nuclear Atox1 are increased after wounding, and that wound healing with and without Cu treatment is impaired in Atox1−/− mice. Endothelial cell (EC)-specific Atox1−/− mice and gene transfer of nuclear-target Atox1 in Atox1−/− mice reveal that Atox1 in ECs as well as transcription factor function of Atox1 are required for wound healing. Mechanistically, Atox1−/− mice show reduced Atox1 target proteins such as p47phox NADPH oxidase and cyclin D1 as well as extracellular matrix Cu enzyme LOX activity in wound tissues. This in turn results in reducing O2− production in ECs, NFkB activity, cell proliferation and collagen formation, thereby inhibiting angiogenesis, macrophage recruitment and extracellular matrix maturation. Our findings suggest that Cu-dependent transcription factor/Cu chaperone Atox1 in ECs plays an important role to sense Cu to accelerate wound angiogenesis and healing.

No MeSH data available.


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Endothelial cell (EC)-specific Atox1−/− mice show delayed wound healing.(A) Strategy to generate EC-specific Atox1−/− mice (EC-Atox1-KO) by crossing Atox1fl/fl with constitutive VE-Cadherin (VEcad)-Cre deleter mice. Exon1-3 of the wild type and recombinant Atox1 locus are shown. The targeting construct included LoxP sites (grey triangles) that flanked exon 2 and a Neo gene cassette flanked by FRT sites (white triangles). The neo gene cassette was removed by crossbreeding with the FLP deleter strain expressing FLP1 recombinase to generate mice with desired floxed allele (Atox1fl). The exon 2-deleted Atox1 allele (Deleted) is depicted following excision by VEcad-Cre recombinase. (B) PCR analysis of genomic DNA isolated from mouse tails. Genotypes for Atox1fl, Atox1+, or VEcad Cre are indicated. (C) Atox1 and α-tubulin (control) protein expression in ECs isolated from mouse hearts (mECs) and aortas (which mainly consists of vascular smooth muscle cells) isolated from Atox1fl/fl (control) and EC-Atox1 KO mice. (D) Representative image of cutaneous wounds (3 mm in diameter) (left panel) and a graph representing mean ± SE of the wound closure rate (right panel) of Atox1fl/fl (control) and EC-Atox1 KO mice after wounding. (n > 3 per group, *p < 0.05 vs. control). Ruler notches 1 mm.
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f6: Endothelial cell (EC)-specific Atox1−/− mice show delayed wound healing.(A) Strategy to generate EC-specific Atox1−/− mice (EC-Atox1-KO) by crossing Atox1fl/fl with constitutive VE-Cadherin (VEcad)-Cre deleter mice. Exon1-3 of the wild type and recombinant Atox1 locus are shown. The targeting construct included LoxP sites (grey triangles) that flanked exon 2 and a Neo gene cassette flanked by FRT sites (white triangles). The neo gene cassette was removed by crossbreeding with the FLP deleter strain expressing FLP1 recombinase to generate mice with desired floxed allele (Atox1fl). The exon 2-deleted Atox1 allele (Deleted) is depicted following excision by VEcad-Cre recombinase. (B) PCR analysis of genomic DNA isolated from mouse tails. Genotypes for Atox1fl, Atox1+, or VEcad Cre are indicated. (C) Atox1 and α-tubulin (control) protein expression in ECs isolated from mouse hearts (mECs) and aortas (which mainly consists of vascular smooth muscle cells) isolated from Atox1fl/fl (control) and EC-Atox1 KO mice. (D) Representative image of cutaneous wounds (3 mm in diameter) (left panel) and a graph representing mean ± SE of the wound closure rate (right panel) of Atox1fl/fl (control) and EC-Atox1 KO mice after wounding. (n > 3 per group, *p < 0.05 vs. control). Ruler notches 1 mm.

Mentions: To demonstrate a specific role of endothelial Atox1 in vivo, we generated EC-specific Atox1 knockout mice (EC-Atox1 KO) by crossing Atox1flox/flox mice with VE-cadherin Cre mice (Fig. 6A,B). ECs isolated from EC-Atox1−/− mice demonstrated loss of Atox1 in ECs compared to those from control Atox1flox/flox mice. By contrast, Atox1 expression in aorta, in which majority of cells are vascular smooth muscle, is not different in both mice (Fig. 6C). These results confirm the EC-specific reduction of Atox1 in vivo. Figure 6D showed that EC-Atox1 KO mice significantly delayed wound closure, suggesting that Atox1 in ECs plays an important role in wound healing.


Endothelial Antioxidant-1: a Key Mediator of Copper-dependent Wound Healing in vivo
Endothelial cell (EC)-specific Atox1−/− mice show delayed wound healing.(A) Strategy to generate EC-specific Atox1−/− mice (EC-Atox1-KO) by crossing Atox1fl/fl with constitutive VE-Cadherin (VEcad)-Cre deleter mice. Exon1-3 of the wild type and recombinant Atox1 locus are shown. The targeting construct included LoxP sites (grey triangles) that flanked exon 2 and a Neo gene cassette flanked by FRT sites (white triangles). The neo gene cassette was removed by crossbreeding with the FLP deleter strain expressing FLP1 recombinase to generate mice with desired floxed allele (Atox1fl). The exon 2-deleted Atox1 allele (Deleted) is depicted following excision by VEcad-Cre recombinase. (B) PCR analysis of genomic DNA isolated from mouse tails. Genotypes for Atox1fl, Atox1+, or VEcad Cre are indicated. (C) Atox1 and α-tubulin (control) protein expression in ECs isolated from mouse hearts (mECs) and aortas (which mainly consists of vascular smooth muscle cells) isolated from Atox1fl/fl (control) and EC-Atox1 KO mice. (D) Representative image of cutaneous wounds (3 mm in diameter) (left panel) and a graph representing mean ± SE of the wound closure rate (right panel) of Atox1fl/fl (control) and EC-Atox1 KO mice after wounding. (n > 3 per group, *p < 0.05 vs. control). Ruler notches 1 mm.
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f6: Endothelial cell (EC)-specific Atox1−/− mice show delayed wound healing.(A) Strategy to generate EC-specific Atox1−/− mice (EC-Atox1-KO) by crossing Atox1fl/fl with constitutive VE-Cadherin (VEcad)-Cre deleter mice. Exon1-3 of the wild type and recombinant Atox1 locus are shown. The targeting construct included LoxP sites (grey triangles) that flanked exon 2 and a Neo gene cassette flanked by FRT sites (white triangles). The neo gene cassette was removed by crossbreeding with the FLP deleter strain expressing FLP1 recombinase to generate mice with desired floxed allele (Atox1fl). The exon 2-deleted Atox1 allele (Deleted) is depicted following excision by VEcad-Cre recombinase. (B) PCR analysis of genomic DNA isolated from mouse tails. Genotypes for Atox1fl, Atox1+, or VEcad Cre are indicated. (C) Atox1 and α-tubulin (control) protein expression in ECs isolated from mouse hearts (mECs) and aortas (which mainly consists of vascular smooth muscle cells) isolated from Atox1fl/fl (control) and EC-Atox1 KO mice. (D) Representative image of cutaneous wounds (3 mm in diameter) (left panel) and a graph representing mean ± SE of the wound closure rate (right panel) of Atox1fl/fl (control) and EC-Atox1 KO mice after wounding. (n > 3 per group, *p < 0.05 vs. control). Ruler notches 1 mm.
Mentions: To demonstrate a specific role of endothelial Atox1 in vivo, we generated EC-specific Atox1 knockout mice (EC-Atox1 KO) by crossing Atox1flox/flox mice with VE-cadherin Cre mice (Fig. 6A,B). ECs isolated from EC-Atox1−/− mice demonstrated loss of Atox1 in ECs compared to those from control Atox1flox/flox mice. By contrast, Atox1 expression in aorta, in which majority of cells are vascular smooth muscle, is not different in both mice (Fig. 6C). These results confirm the EC-specific reduction of Atox1 in vivo. Figure 6D showed that EC-Atox1 KO mice significantly delayed wound closure, suggesting that Atox1 in ECs plays an important role in wound healing.

View Article: PubMed Central - PubMed

ABSTRACT

Copper (Cu), an essential nutrient, promotes wound healing, however, target of Cu action and underlying mechanisms remain elusive. Cu chaperone Antioxidant-1 (Atox1) in the cytosol supplies Cu to the secretory enzymes such as lysyl oxidase (LOX), while Atox1 in the nucleus functions as a Cu-dependent transcription factor. Using mouse cutaneous wound healing model, here we show that Cu content (by X-ray Fluorescence Microscopy) and nuclear Atox1 are increased after wounding, and that wound healing with and without Cu treatment is impaired in Atox1&minus;/&minus; mice. Endothelial cell (EC)-specific Atox1&minus;/&minus; mice and gene transfer of nuclear-target Atox1 in Atox1&minus;/&minus; mice reveal that Atox1 in ECs as well as transcription factor function of Atox1 are required for wound healing. Mechanistically, Atox1&minus;/&minus; mice show reduced Atox1 target proteins such as p47phox NADPH oxidase and cyclin D1 as well as extracellular matrix Cu enzyme LOX activity in wound tissues. This in turn results in reducing O2&minus; production in ECs, NFkB activity, cell proliferation and collagen formation, thereby inhibiting angiogenesis, macrophage recruitment and extracellular matrix maturation. Our findings suggest that Cu-dependent transcription factor/Cu chaperone Atox1 in ECs plays an important role to sense Cu to accelerate wound angiogenesis and healing.

No MeSH data available.


Related in: MedlinePlus