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First published online December 11, 2006
doi: 10.1242/10.1242/jcs.03320

Journal of Cell Science 119, 5193-5203 (2006)
Published by The Company of Biologists 2006
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Acute downregulation of connexin43 at wound sites leads to a reduced inflammatory response, enhanced keratinocyte proliferation and wound fibroblast migration

Ryoichi Mori1, Kieran T. Power3,*, Chiuhui Mary Wang3,*, Paul Martin1,2 and David L. Becker3,{ddagger}

1 Department of Physiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
2 Department of Biochemistry, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK
3 Department of Anatomy and Developmental Biology, University College of London, Gower Street, London, WC1E 6BT, UK


Figure 1
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  		Fig. 1. Expression of Cx43 at wound sites. (A) Real-time PCR analysis of the gene expression of Cx43 at wound sites. Relative expression levels of Cx43 to GAPDH on days 1 and 7 in wounds treated with control sODN (n=4; white bars) and Cx43-asODN (n=4; black bars). Data are expressed as the mean ± s.e.m.; *P<0.05. (B-F) Cx43 staining (green) with bis-benzimide nuclear staining (blue) of control wounds (B, 1 day; D, 2 days; F, 7 days) or Cx43-asODN-treated wounds (C, 1 day; E, 2 days; G, 7 days). (H,I) Illustration of sites imaged in the wound edge. (H) Images B-E. (I) Images F and G. e, epidermis; d, dermis. For a high-resolution figure, please see the online version of this article.

 

Figure 2
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  		Fig. 2. Cell proliferation. (A-D) Analysis of cell proliferation at wound sites by immunohistochemical staining with the anti-BrdU monoclonal antibody in control ODN (A, day 2; C, day 7) and Cx43-asODN (B, day 2; D, day 7). Arrowhead and arrow indicate the wound margin and leading edge, respectively. (E,F) The number of BrdU-positive cells per field in the wound margin in the epidermis (E; n=5) and the nascent epidermis (F; n=5). (G,H) The number of BrdU-positive cells per 1.33 mm2 in the dermal wound edge (G; n=5) and in the forming granulation tissue (H; n=5). Counts are expressed as the mean ± s.e.m.; *P<0.05. Bars, 200 µm.

 

Figure 3
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  		Fig. 3. Neutrophil recruitment into the wound site. (A,B) Neutrophil recruitment into skin wounds treated with control sODN (A) and Cx43-asODN (B), analyzed using an anti-MPO antibody on day 1. (C) Numbers of MPO-positive cells per 0.332 mm2 at the wound site after treatment with control sODN (white bars: n=4 on day 1; n=5 on day 2) and Cx43-asODN (black bars; n=3 on day 1; n=4 on day 2). Data are expressed as the mean ± s.e.m. *P<0.05. Bars, 50 µm.

 

Figure 4
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  		Fig. 4. Macrophage recruitment into the wound site. (A,B) Macrophage recruitment into skin wounds treated with control sODN (A) and Cx43-asODN (B), analyzed using an anti-F4/80 antibody on day 7. (C) Macrophage recruitment into skin wounds, cells per 0.332 mm2 on days 2 and 7 after treatment with control sODN (white bars: n=4 on day 2, n=7 on day 7) and Cx43-asODN (black bars: n=4 on day 2, n=6 on day 7). Data are expressed as the mean ± s.e.m.; **P<0.01. Bars, 50 µm.

 

Figure 5
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  		Fig. 5. Expression of Ccl2 and TNF-{alpha} at wound sites. (A,B) Real-time PCR analysis of the gene expression of Ccl2 and TNF-{alpha} at wound sites. Relative expression levels of Ccl2 (A) and TNF-{alpha} (B) to GAPDH on days 1, 2 and 7 (n=5 for each) after treatment with control sODN (white bars) or Cx43-asODN (black bars). Data are expressed as the mean ± s.e.m.; *P<0.05.

 

Figure 6
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  		Fig. 6. Expression of TGF-ß1. (A-J) Immunohistochemistry for TGF-ß1 at wound sites treated with control sODN (A-E) and Cx43-asODN (F-J). Bars, 200 µm (A,F) and 50 µm (B-E,G-J). Black arrows show the nascent edge of the epidermis. TGF-ß1 staining is considerably stronger in the epidermis of wounds treated with Cx43-asODN (I,J) compared with those treated with control sODN (D,E). Red and black arrowheads indicate representative TGF-ß1-elongated fibroblast-like cells and rounded presumptive leukocytes, respectively. (K) Real-time PCR analysis of the expression on days 1, 2 and 7 (n=5 for each) of mRNA for TGF-ß1 at wound sites treated with control sODN (white bars) or Cx43-asODN (black bars). Data are expressed as the mean ± s.e.m.; *P<0.05.

 

Figure 7
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  		Fig. 7. Granulation tissue formation and fibroblast migration. (A,B) Fibroblast-like cell recruitment into skin wounds treated with control sODN (A) and Cx43-asODN (B), analyzed using TRITC-phalloidin and DAPI nuclear staining on day 2. (C) Number of fibroblast-like cells at each wound site per field of view for wounds treated with control sODN (white bars, n=5) or Cx43-asODN (black bars, n=5). (D-E) Images of wounds in fibroblast cultures at (D) the time of wounding and (E) 4 hours after wounding. (F) Wound-healing assay of fibroblast migration shows that migration is significantly faster after treatment with Cx43-asODNs. (G,H) Staining of fibroblasts for Cx43 in cultures treated with (G) control and (H) Cx43-asODN after 2.5 hours. (I) Quantification of Cx43 expression 2.5 hours after Cx43-asODN treatment, expressed as a percentage of control (n=8 per time point). Data are expressed as the mean ± s.e.m.; *P<0.02, **P<0.01, ***P<0.0001. Bars, 50 µm. For a high-resolution figure, please see the online version of this article.

 

Figure 8
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  		Fig. 8. Collagen expression in the wound site. (A) Collagen content was assessed by quantitatively measuring the hydroxyproline (HP) content on days 7, 10 and 14 after wounding at wound sites treated with control sODN (white bars) and Cx43-asODN (black bars) and in uninjured skin (n=5). Data are expressed as the mean ± s.e.m. P<0.05. (B) Real-time PCR analysis of the expression of mRNA on days 2 and 7 (n=5 for each) for Col1{alpha}1 at wound sites treated with control sODN (white bars) and Cx43-asODN (black bars). Data are expressed as the mean ± s.e.m.; *P<0.05.

 

Figure 9
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  		Fig. 9. Granulation tissue contraction. (A,B) H and E staining of 14-days-old wound granulation tissue in wounds treated with control sODN (A) and asODN (B). (C) Area of granulation tissue after treatment with control sODN (white bars) or Cx43-asODN (black bars) analyzed on day 5 (control, n=7; asODN, n=6), day 7 (control, n=5; asODN, n=5), day 10 (control, n=5; asODN, n=6), and day 14 (control, n=5; asODN, n=6). Granulation tissue area measurements at day 5 already showed a slightly smaller area after treatment but the reduction became significant on days 7, 10 and 14 (*P<0.05, **P<0.01). Data are expressed as the mean ± s.e.m. Bars, 1 mm.

 

Figure 10
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  		Fig. 10. Apoptosis. (A, B) TUNEL staining of granulation tissue in (A) control sODN and (B) Cx43-asODN-treated wounds on day 7. Apoptotic cells appear as bright green spots, some of which have been highlighted with arrowheads. (C) Numbers of apoptotic cells per field of view on days 5, 7 and 10 (n=6 for each) in wound sites treated with control sODN (white bars) and Cx43-asODN (black bars). Data are expressed as the mean ± s.e.m. Bars, 50 µm. For a high-resolution figure, please see the online version of this article.

 

Figure 11
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  		Fig. 11. Myofibroblast maturation. (A-D) anti-{alpha}SMA antibody staining (green) with bis-benzimide nuclear staining (blue) of (A,B) the edge of the granulation tissue in a 7 day wound and (C,D) the center of the granulation tissue in a 10 day wound. (E) Quantification of staining levels showed significantly more {alpha}-SMA staining in wounds treated with Cx43-asODN than with control sODN at 7 days (P=0.004) indicating earlier maturation and differentiation of myofibroblasts. This more advanced maturation was still present at 10 days when most of the {alpha}-SMA staining and myofibroblasts were lost in wounds treated with Cx43-asODN, but staining was still very strong in control wounds (P=0.000002). (F) Illustration of sites imaged in the granulation tissue. Zone I (A,B) zone II (C,D). e, epidermis; d, dermis. Data are expressed as the mean ± s.e.m. Bars, 25 µm. For a high-resolution figure, please see the online version of this article.

 

Figure 12
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  		Fig. 12. Angiogenesis. (A-F) von Willebrand factor staining of nacent blood vessels granulation tissue (green) with bis-benzimide nuclear stain (blue) at 7 days (A,B), 10 days (C,D) and 14 days (E,F) after wounding. In Cx43-asODN-treated wounds (B,D,F) blood vessels were more pervasive at early time points (7 days, B; 10 days, D) but considerably finer than those of controls (A,C,E) resulting in significantly reduced staining compared with controls (7 days *P=0.0019; 10 days **P=0.015). By 14 days, blood vessels had increased in size in the asODN group and were a similar size to those of controls (G). (H) Illustration of sites imaged in the granulation tissue. Zone I (A,B) zone II (C-E). e, epidermis; d, dermis. Per treatment and time point six to eight animals were used. Data are expressed as the mean ± s.e.m. Bars, 25 µm. For a high-resolution figure, please see the online version of this article.

 

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© The Company of Biologists Ltd 2006