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Resistance of keratinocytes to TGFß-mediated growth restriction and apoptosis induction accelerates re-epithelialization in skin wounds

Christiane Amendt1, Amrit Mann1, Peter Schirmacher2 and Manfred Blessing1,*

1 I. Medical Department, Section Pathophysiology, Johannes Gutenberg-University, D-55131 Mainz, Germany
2 Institute of Pathology, University of Cologne, D-50931 Cologne, Germany



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  		Fig. 1. Re-epithelialization of full thickness excisional wounds of wild-type and transgenic mice at day 11 after wounding. (A) Hematoxylin-Eosin stained cryosection through the mid-point of a wound from a wild-type animal showing an incomplete re-epithelialization. The wound edges were marked by arrow heads. (B) Section through the mid-point of a wound from a transgenic animal where the re-epithelialization process is already completed. Bars, 100 µm.

 


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  		Fig. 2. Hematopoietic cells in the granulation tissue. Giemsa stained cryosections of 10-day-old wounds (A,B) and 14-day-old wounds (C,D). At day 10, the number of giemsa stained cells is similar in both transgenic (A) and non-transgenic (B) animals, whereas at day 14, the number of giemsa stained cells is clearly reduced in transgenic animals (C) in comparison with non-transgenic animals (D). Arrowheads indicate newly formed epidermis; arrows indicate hematopoietic cells. Bars, 50 µm. (E) Quantification of hematopoietic cells. Counting of giemsa stained cells in 64 mm2 fields of a photograph from wounds of at least four different animals revealed a significantly reduced number in transgenic animals compared with non-transgenic animals at day 14 after wounding (P<0.02). Error bars represent the standard deviation.

 


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  		Fig. 3. Characterization of hematopoietic cells in the granulation tissue. Immunohistochemistry using Mac1 (CD11b)-antibody for detection of macrophages (A,B), and naphthol-ASD-chloroacetate-esterase histochemistry for detection of neutrophils and mast cells (C,D). Transgenic mice (A,C) exhibited significantly fewer numbers of macrophages (A), as well as neutrophils and mast cells (C) in cryosections of 14-day-old wounds when compared with the non-transgenic controls (B,D, respectively). Arrows indicate macrophages (A,B), and mast cells or neutrophils (C,D); arrowheads indicate the epidermal/dermal junction (A-D). Bars, 50 µm.

 


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  		Fig. 4. Keratinocyte proliferation during wound healing. (A-F) At least three mice of every group were injected intraperitoneally with BrdU and sacrificed after a labeling period of 1 hour. Skin sections were stained for detection of BrdU-labeled S-phase nuclei (green, arrows) and for keratin 14 marking the epidermis (red). Transgenic animals (A,C,E) exhibit a higher number of labeled nuclei at the wound edge at 3, 5 and 7 days post wounding compared with wild-type animals (B,D,F). 3 days post wounding (A,B); 5 days post wounding (C,D); and 7 days post wounding (E,F). Bars, 50 µm.

 


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  		Fig. 5. Apoptosis in the epidermis of 13-day-old wounds. (A,C) Apoptotic cells in the newly formed epidermis, which is stained by an antikeratin 14 antibody (red), were marked by TUNEL-labeling (green; arrow heads). (B,D) All cell nuclei were stained with DAPI (blue). In non-transgenic animals (A,B), a significantly higher number of apoptotic cells were detected compared with transgenic animals (C,D). (E) The percentage of apoptotic cells. Only 0.42±0.16% basal epidermal cells in transgenic animals undergo apoptosis, whereas in wild-type animals 0.77±0.10% basal apoptotic cells are found. Non-transgenic animals, n=5; transgenic animals, n=4; *P<0.005. Bars, 50 µm.

 


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  		Fig. 6. Expression of Egr1 in wounds. (A) A cDNA-array was probed with cDNA derived from 7-day-old wounds of transgenic and non-transgenic animals. Egr1 expression (arrows) was reduced 7 times in transgenic animals compared with non-transgenic animals. (B) Northern blot analysis for Egr1 using RNA form 13-day-old wounds. In normal skin, Egr1 expression (3.2 kb) is low, whereas in 13-day-old wounds Egr1 expression is elevated in comparison with unwounded skin only in non-transgenic animals. GAPDH expression (1.3 kb) was used as loading control. (C) Quantification of the northern blot analysis by PhosphorImager system. In 13-day-old wounds, the expression level of Egr1 is threefold higher in non-transgenic animals in comparison with unwounded skin. By contrast, no upregulation is found in transgenic animals.

 


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  		Fig. 7. Immunodetection of Egr1 in wounds. Cryosections of 13-day-old wounds from wild-type animals were stained with an antibody against Egr1. Keratinocytes in the newly formed epithelium at the wound margin exhibit strong induction of Egr1 (A), whereas distal to the wound margin, hardly any upregulation is seen (B). Arrowheads indicates the epidermal/dermal junction. Bars, 50 µm.

 


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  		Fig. 8. Expression of Egr1 in wild-type HaCaT cells and HaCaT cells transfected with an expression vector for the dominant-negative type II TGFß receptor. (A) Expression of the dominant negative type II TGFß receptor in two transfected HaCaT clones. The northern blot was probed with cDNA of the dnTGFßRII and with keratin 5 cDNA. Lane 1 shows HaCaT clone dn, which has a high expression level of the dnTGFßRII (2.4 kb). Lane 2 shows the very low expressing clone neo. Keratin 5 expression (2.6 kb) was used as loading control. (B) Northern blot analysis of RNA from HaCaT cells transfected with the dominant-negative type II TGFß receptor (wt) and transfectants (dn). Without addition of TGFß1, the expression of Egr1 in wild-type and transfectant cells is very low. Forty-five minutes after addition of 5 ng/ml TGFß1, the expression of Egr1 (3.2 kb) is strongly induced in untransfected HaCaT cells only. GAPDH expression (1.3 kb) was used as loading control. (C) Quantification of the northern blot analysis by PhosphorImager system. The expression level of Egr1 after addition of 5 ng/ml TGFß1 (+TGFß1) is elevated 24-fold in HaCaT cells and neo controls. By contrast, in HaCaT cells expressing the dominant-negative type II receptor at high levels (dn), only a ninefold increase is found.

 

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