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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.


Related in: MedlinePlus

Atox1 is expressed in endothelial and inflammatory cells in the dermis during wound healing.(A,B) Hematoxylin & Eosin (H&E) and representative immunostaining for Atox1 (green), Mac3 (red, macrophage marker), TO-PRO3 (blue, nuclear marker) or their colocalization (Merge) on day 5 (A); and Atox1 (red), CD31 (green, EC marker), TO-PRO3 (blue, nuclear marker) or their colocalization (Merge) on day 7 (B) in the dermal region of wound tissues (W). Serial section stained with H&E (A,B), W: wound area; WE: wound edge, Scale bars = 10 μm.
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f4: Atox1 is expressed in endothelial and inflammatory cells in the dermis during wound healing.(A,B) Hematoxylin & Eosin (H&E) and representative immunostaining for Atox1 (green), Mac3 (red, macrophage marker), TO-PRO3 (blue, nuclear marker) or their colocalization (Merge) on day 5 (A); and Atox1 (red), CD31 (green, EC marker), TO-PRO3 (blue, nuclear marker) or their colocalization (Merge) on day 7 (B) in the dermal region of wound tissues (W). Serial section stained with H&E (A,B), W: wound area; WE: wound edge, Scale bars = 10 μm.

Mentions: To gain insight into the mechanisms by which Atox1 is involved in wound healing which is dependent on inflammation at early phase and angiogenesis at later phase152930 we performed immunofluorescence analysis to examine the expression and localization of Atox1 protein in skin tissues after wounding. Atox1 was highly expressed in Mac3+ macrophage at day 5 (Fig. 4A) and in capillary-like CD31+ ECs at day 7 (Fig. 4B). Of note, some Atox1 positive cells in ECs and macrophages were found in the nucleus. We then examined the functional role of Atox1 in wound-induced angiogenesis and inflammatory cell recruitment, which are involved in wound healing. Immunohistological and laser Doppler flow analysis demonstrated that wounding-induced increase in the number of capillary-like CD31+ ECs (Fig. 5A) and blood flow perfusion (Fig. 5B) in wound tissues were significantly inhibited in Atox1−/− mice. Moreover, Atox1−/− mice showed decreased number of infiltrated Mac3+ macrophage in wounded tissues (Fig. 5C), which was associated with reduced expression of chemokine SDF1α and VCAM1 (Fig. 5D) which was shown to be secreted by macrophages to promote revascularization and tissue repair313233. These results suggest that Atox1 is required for wound healing by regulating angiogenesis and inflammatory cell recruitment to the injured sites.


Endothelial Antioxidant-1: a Key Mediator of Copper-dependent Wound Healing in vivo
Atox1 is expressed in endothelial and inflammatory cells in the dermis during wound healing.(A,B) Hematoxylin & Eosin (H&E) and representative immunostaining for Atox1 (green), Mac3 (red, macrophage marker), TO-PRO3 (blue, nuclear marker) or their colocalization (Merge) on day 5 (A); and Atox1 (red), CD31 (green, EC marker), TO-PRO3 (blue, nuclear marker) or their colocalization (Merge) on day 7 (B) in the dermal region of wound tissues (W). Serial section stained with H&E (A,B), W: wound area; WE: wound edge, Scale bars = 10 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5036036&req=5

f4: Atox1 is expressed in endothelial and inflammatory cells in the dermis during wound healing.(A,B) Hematoxylin & Eosin (H&E) and representative immunostaining for Atox1 (green), Mac3 (red, macrophage marker), TO-PRO3 (blue, nuclear marker) or their colocalization (Merge) on day 5 (A); and Atox1 (red), CD31 (green, EC marker), TO-PRO3 (blue, nuclear marker) or their colocalization (Merge) on day 7 (B) in the dermal region of wound tissues (W). Serial section stained with H&E (A,B), W: wound area; WE: wound edge, Scale bars = 10 μm.
Mentions: To gain insight into the mechanisms by which Atox1 is involved in wound healing which is dependent on inflammation at early phase and angiogenesis at later phase152930 we performed immunofluorescence analysis to examine the expression and localization of Atox1 protein in skin tissues after wounding. Atox1 was highly expressed in Mac3+ macrophage at day 5 (Fig. 4A) and in capillary-like CD31+ ECs at day 7 (Fig. 4B). Of note, some Atox1 positive cells in ECs and macrophages were found in the nucleus. We then examined the functional role of Atox1 in wound-induced angiogenesis and inflammatory cell recruitment, which are involved in wound healing. Immunohistological and laser Doppler flow analysis demonstrated that wounding-induced increase in the number of capillary-like CD31+ ECs (Fig. 5A) and blood flow perfusion (Fig. 5B) in wound tissues were significantly inhibited in Atox1−/− mice. Moreover, Atox1−/− mice showed decreased number of infiltrated Mac3+ macrophage in wounded tissues (Fig. 5C), which was associated with reduced expression of chemokine SDF1α and VCAM1 (Fig. 5D) which was shown to be secreted by macrophages to promote revascularization and tissue repair313233. These results suggest that Atox1 is required for wound healing by regulating angiogenesis and inflammatory cell recruitment to the injured sites.

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.


Related in: MedlinePlus