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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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Exogenous and endogenous Cu promote wound healing.(A) Excisional cutaneous wounds were created using a 6 mm biopsy punch on the dorsal skin of WT mice, and treated with Cu (1.25 nmoles) or Cu chelator BCS (10 nmoles), or vehicle (PBS). Ruler notches, 1 mm. A graph represents the mean ± SE of wound closure rates (*p < 0.05; #p < 0.05 vs PBS). (B) Expression of Cu chaperones, including Atox1, CCS, and Cox17, and α-tubulin (loading control) in wound tissues after injury. A graph represents mean ± SE at indicated date post-wounding (n = 3 at each time, *p < 0.001 vs. day 0).
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f1: Exogenous and endogenous Cu promote wound healing.(A) Excisional cutaneous wounds were created using a 6 mm biopsy punch on the dorsal skin of WT mice, and treated with Cu (1.25 nmoles) or Cu chelator BCS (10 nmoles), or vehicle (PBS). Ruler notches, 1 mm. A graph represents the mean ± SE of wound closure rates (*p < 0.05; #p < 0.05 vs PBS). (B) Expression of Cu chaperones, including Atox1, CCS, and Cox17, and α-tubulin (loading control) in wound tissues after injury. A graph represents mean ± SE at indicated date post-wounding (n = 3 at each time, *p < 0.001 vs. day 0).

Mentions: Cu treatment accelerates wound closure and healing; however, underlying mechanism and role of endogenous Cu remains elusive5679111213. To address this question, we made four excisional wounds on the dorsal skin in WT mice. Topical Cu treatment of the wound site significantly accelerated wound contraction and closures whereas Cu chelator BCS retarded the healing process (Fig. 1A). Figure 1B showed that wounding markedly increased expression of Cu chaperone Atox1, but not other chaperones such as CCS or Cox17 in a time-dependent manner. To determine the functional role of Atox1 in wound healing in vivo, we used Atox1−/− mice and WT mice. Histological and quantitative morphometric analysis revealed that Atox1−/− mice exhibited a marked decrease in wound closure rate as well as more rapid epithelialization by histological analysis (Fig. 2A). Figure 2B,C showed Cu-induced enhancement of wound healing as well as an increase in Atox1 (data not shown) and VEGF protein expression induced by wounding were markedly decreased in Atox1−/− mice. These findings indicate that Atox1 senses Cu to promote wound healing at least via regulating VEGF expression, which has been shown to be essential for wound repair26.


Endothelial Antioxidant-1: a Key Mediator of Copper-dependent Wound Healing in vivo
Exogenous and endogenous Cu promote wound healing.(A) Excisional cutaneous wounds were created using a 6 mm biopsy punch on the dorsal skin of WT mice, and treated with Cu (1.25 nmoles) or Cu chelator BCS (10 nmoles), or vehicle (PBS). Ruler notches, 1 mm. A graph represents the mean ± SE of wound closure rates (*p < 0.05; #p < 0.05 vs PBS). (B) Expression of Cu chaperones, including Atox1, CCS, and Cox17, and α-tubulin (loading control) in wound tissues after injury. A graph represents mean ± SE at indicated date post-wounding (n = 3 at each time, *p < 0.001 vs. day 0).
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036036&req=5

f1: Exogenous and endogenous Cu promote wound healing.(A) Excisional cutaneous wounds were created using a 6 mm biopsy punch on the dorsal skin of WT mice, and treated with Cu (1.25 nmoles) or Cu chelator BCS (10 nmoles), or vehicle (PBS). Ruler notches, 1 mm. A graph represents the mean ± SE of wound closure rates (*p < 0.05; #p < 0.05 vs PBS). (B) Expression of Cu chaperones, including Atox1, CCS, and Cox17, and α-tubulin (loading control) in wound tissues after injury. A graph represents mean ± SE at indicated date post-wounding (n = 3 at each time, *p < 0.001 vs. day 0).
Mentions: Cu treatment accelerates wound closure and healing; however, underlying mechanism and role of endogenous Cu remains elusive5679111213. To address this question, we made four excisional wounds on the dorsal skin in WT mice. Topical Cu treatment of the wound site significantly accelerated wound contraction and closures whereas Cu chelator BCS retarded the healing process (Fig. 1A). Figure 1B showed that wounding markedly increased expression of Cu chaperone Atox1, but not other chaperones such as CCS or Cox17 in a time-dependent manner. To determine the functional role of Atox1 in wound healing in vivo, we used Atox1−/− mice and WT mice. Histological and quantitative morphometric analysis revealed that Atox1−/− mice exhibited a marked decrease in wound closure rate as well as more rapid epithelialization by histological analysis (Fig. 2A). Figure 2B,C showed Cu-induced enhancement of wound healing as well as an increase in Atox1 (data not shown) and VEGF protein expression induced by wounding were markedly decreased in Atox1−/− mice. These findings indicate that Atox1 senses Cu to promote wound healing at least via regulating VEGF expression, which has been shown to be essential for wound repair26.

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