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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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Atox1 is involved in p47phox-ROS-NFkB activation as well as cyclin D1-cell proliferation in wound tissue.(A) p47phox and α-tubulin (loading control) protein expression in wound tissues at day 7 in WT and Atox1−/− mice. (B) Representative images for CD31 staining (green), DHE fluorescence (red), and their merged images (yellow) in wound tissues at day 7 after wounding in WT mice. DHE+/CD31+ ECs (yellow) are shown in white arrows. Right panel shows mean ± SE of CD31+/DHE+ cells in WT and Atox1−/− mice (n = 3, *p < 0.05 vs. WT). Scale bars = 10 μm (C) Representative bioluminescence images of back skin of NFkB activity reporter mice (HLL mice) and HLL/Atox1 KO mice before and at 7 day after wounding (n = 3)(left) and a graph representing mean ± SE of bioluminescence intensity (n = 3, *p < 0.05 vs. HLL). (D,E) Cell proliferation in wounds were assessed by Cyclin D1 staining, Scale bar = 50 μm (left images); scale bar = 10 μm (right images). (D) and BrdU with DAPI (blue, nuclear marker) staining (E) at day 5 after wounding in WT and Atox1−/− mice. Graphs represents mean ± SE of cyclin D1+ cells and BrdU+ cells (n = 3, *p < 0.05; **p < 0.01 vs WT) (F) Immunofluorescence showing co-localization of BrdU+ cells (red) and Atox1 (Green) in the nucleus in dermal region at day 5 after wounding in WT mice. Scale bars = 10 μm.
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f8: Atox1 is involved in p47phox-ROS-NFkB activation as well as cyclin D1-cell proliferation in wound tissue.(A) p47phox and α-tubulin (loading control) protein expression in wound tissues at day 7 in WT and Atox1−/− mice. (B) Representative images for CD31 staining (green), DHE fluorescence (red), and their merged images (yellow) in wound tissues at day 7 after wounding in WT mice. DHE+/CD31+ ECs (yellow) are shown in white arrows. Right panel shows mean ± SE of CD31+/DHE+ cells in WT and Atox1−/− mice (n = 3, *p < 0.05 vs. WT). Scale bars = 10 μm (C) Representative bioluminescence images of back skin of NFkB activity reporter mice (HLL mice) and HLL/Atox1 KO mice before and at 7 day after wounding (n = 3)(left) and a graph representing mean ± SE of bioluminescence intensity (n = 3, *p < 0.05 vs. HLL). (D,E) Cell proliferation in wounds were assessed by Cyclin D1 staining, Scale bar = 50 μm (left images); scale bar = 10 μm (right images). (D) and BrdU with DAPI (blue, nuclear marker) staining (E) at day 5 after wounding in WT and Atox1−/− mice. Graphs represents mean ± SE of cyclin D1+ cells and BrdU+ cells (n = 3, *p < 0.05; **p < 0.01 vs WT) (F) Immunofluorescence showing co-localization of BrdU+ cells (red) and Atox1 (Green) in the nucleus in dermal region at day 5 after wounding in WT mice. Scale bars = 10 μm.

Mentions: We previously reported that Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox in ECs25 as well as cyclin D1 in mouse fibroblasts24. This is further supported by findings that nuclear-targeted Atox1 increases cyclin D1 and p47phox expression in ECs (supplemental Fig. 1). Consistently, wound-induced increase in p47phox expression (Fig. 8A) and O2− production in ECs (DHE+/CD31+ cells)(Fig. 8B) in wound tissues were significantly inhibited in Atox1−/− mice at day7 after wounding when contribution of inflammatory cells is minor. Moreover, in vivo Bioluminescence imaging of NF-kB reporter (HLL) mice showed that wound injury-induced increase in redox-sensitive NF-kB activity was markedly decreased in HLL mice crossed with Atox1−/− mice (Fig. 8C). Moreover, we found that cyclin D1 + cells (Fig. 8D) as well as BrdU+ cells (Fig. 8E), which partially colocalized with Atox1+ cells (Fig. 8F) in the nucleus within dermis region in wound tissues, were significantly decreased in Atox1−/− mice. These findings suggest that Atox1 is involved in ROS production and its downstream NF-kB activity via upregulating p47phox as well as cell proliferation via upregulating cyclin D1, thereby promoting wound healing.


Endothelial Antioxidant-1: a Key Mediator of Copper-dependent Wound Healing in vivo
Atox1 is involved in p47phox-ROS-NFkB activation as well as cyclin D1-cell proliferation in wound tissue.(A) p47phox and α-tubulin (loading control) protein expression in wound tissues at day 7 in WT and Atox1−/− mice. (B) Representative images for CD31 staining (green), DHE fluorescence (red), and their merged images (yellow) in wound tissues at day 7 after wounding in WT mice. DHE+/CD31+ ECs (yellow) are shown in white arrows. Right panel shows mean ± SE of CD31+/DHE+ cells in WT and Atox1−/− mice (n = 3, *p < 0.05 vs. WT). Scale bars = 10 μm (C) Representative bioluminescence images of back skin of NFkB activity reporter mice (HLL mice) and HLL/Atox1 KO mice before and at 7 day after wounding (n = 3)(left) and a graph representing mean ± SE of bioluminescence intensity (n = 3, *p < 0.05 vs. HLL). (D,E) Cell proliferation in wounds were assessed by Cyclin D1 staining, Scale bar = 50 μm (left images); scale bar = 10 μm (right images). (D) and BrdU with DAPI (blue, nuclear marker) staining (E) at day 5 after wounding in WT and Atox1−/− mice. Graphs represents mean ± SE of cyclin D1+ cells and BrdU+ cells (n = 3, *p < 0.05; **p < 0.01 vs WT) (F) Immunofluorescence showing co-localization of BrdU+ cells (red) and Atox1 (Green) in the nucleus in dermal region at day 5 after wounding in WT mice. Scale bars = 10 μm.
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Related In: Results  -  Collection

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f8: Atox1 is involved in p47phox-ROS-NFkB activation as well as cyclin D1-cell proliferation in wound tissue.(A) p47phox and α-tubulin (loading control) protein expression in wound tissues at day 7 in WT and Atox1−/− mice. (B) Representative images for CD31 staining (green), DHE fluorescence (red), and their merged images (yellow) in wound tissues at day 7 after wounding in WT mice. DHE+/CD31+ ECs (yellow) are shown in white arrows. Right panel shows mean ± SE of CD31+/DHE+ cells in WT and Atox1−/− mice (n = 3, *p < 0.05 vs. WT). Scale bars = 10 μm (C) Representative bioluminescence images of back skin of NFkB activity reporter mice (HLL mice) and HLL/Atox1 KO mice before and at 7 day after wounding (n = 3)(left) and a graph representing mean ± SE of bioluminescence intensity (n = 3, *p < 0.05 vs. HLL). (D,E) Cell proliferation in wounds were assessed by Cyclin D1 staining, Scale bar = 50 μm (left images); scale bar = 10 μm (right images). (D) and BrdU with DAPI (blue, nuclear marker) staining (E) at day 5 after wounding in WT and Atox1−/− mice. Graphs represents mean ± SE of cyclin D1+ cells and BrdU+ cells (n = 3, *p < 0.05; **p < 0.01 vs WT) (F) Immunofluorescence showing co-localization of BrdU+ cells (red) and Atox1 (Green) in the nucleus in dermal region at day 5 after wounding in WT mice. Scale bars = 10 μm.
Mentions: We previously reported that Atox1 functions as a Cu-dependent transcription factor for NADPH oxidase organizer p47phox in ECs25 as well as cyclin D1 in mouse fibroblasts24. This is further supported by findings that nuclear-targeted Atox1 increases cyclin D1 and p47phox expression in ECs (supplemental Fig. 1). Consistently, wound-induced increase in p47phox expression (Fig. 8A) and O2− production in ECs (DHE+/CD31+ cells)(Fig. 8B) in wound tissues were significantly inhibited in Atox1−/− mice at day7 after wounding when contribution of inflammatory cells is minor. Moreover, in vivo Bioluminescence imaging of NF-kB reporter (HLL) mice showed that wound injury-induced increase in redox-sensitive NF-kB activity was markedly decreased in HLL mice crossed with Atox1−/− mice (Fig. 8C). Moreover, we found that cyclin D1 + cells (Fig. 8D) as well as BrdU+ cells (Fig. 8E), which partially colocalized with Atox1+ cells (Fig. 8F) in the nucleus within dermis region in wound tissues, were significantly decreased in Atox1−/− mice. These findings suggest that Atox1 is involved in ROS production and its downstream NF-kB activity via upregulating p47phox as well as cell proliferation via upregulating cyclin D1, thereby promoting 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