1. Expression and Activation of Matrix Metalloproteinases in Wounds: Modulation by the Tripeptide–Copper Complex Glycyl-L-Histidyl-L-Lysine-Cu2+ 
Alain Siméon, Frédérique Monier, Hervé Emonard, Philippe Gillery, William Hornebeck, François-Xavier Maquart 
Journal of Investigative Dermatology 
Volume 112, Issue 6, Pages (June 1999)
DOI: /j x
Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
GHK-Cu injections accelerates the healing process. Histologic examination of the chamber content at day 7 (A, B) and day 21 (C, D) after implantation. At day 7, more inflammatory cells were present in the treated chambers (B) than in controls (A). At day 21, wound tissue of the GHK-Cu injected wound chambers is organized in large and dense fibrosis (C), whereas controls exhibit more edematous areas (D). Scale bar: 50 μm.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
GHK-Cu injections increase extracellular matrix accumulation in wound chambers. Chambers were injected twice a week without (□, controls) or with GHK-Cu, 2.0 mg per injection (▪). Rats were killed and the chamber content collected for analysis on days 7, 12, 18, or 22. (A) Dry weight; (B) DNA; (C) total proteins; (D) collagen hydroxyproline; (E) hydroxyproline from the small peptides. Results were expressed as mean of four rats ± SEM. *Significantly different from the corresponding control, p < 0.05, **p < 0.01.
Journal of Investigative Dermatology  , DOI: ( /j x)
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4. Figure 3
MMP-2 and MMP-9 are the main gelatinases expressed during wound healing. Gelatinolytic activity in wound fluid (A) and wound tissue (B) collected from a wound chamber injected with 2 mg GHK-Cu (+) or with saline (controls, –) twice a week from day 0 to day 21. The day of sample collection for analysis is indicated. Samples were analyzed by zymography in gelatin-containing gels. Ten micrograms of protein were applied on each lane.
Journal of Investigative Dermatology  , DOI: ( /j x)
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5. Figure 4
Identification of the 95 kDa gelatinase as proMMP-9. Zymographic analysis of wound fluid collected 3 d after wound chamber implantation. (A) Inhibition of the gelatinase activity by the addition of 10 mM EDTA in the incubation buffer (lane 2) vs. control without EDTA (lane 1). (B) Samples were incubated with (lane 4) or without (lane 3) 1 mM AMPA for 2 h at 37°C to study MMP activation.
Journal of Investigative Dermatology  , DOI: ( /j x)
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6. Figure 5
Rapid decrease of proMMP-9 expression in wound fluid (A) and tissue (B). Densitometric analysis of the zymograms on a Desaga densitometer demonstrated that proMMP-9 was highly expressed at the early times of the wound healing process then decreased rapidly. GHK-Cu injections induced an increased pro-MMP9 expression in the wound tissue on days 18 and 22 only. Every data represents the mean of three experiments ± 1 SEM. ▪, controls; ○, GHK-Cu-injected rats. *Significantly different from the corresponding controls, p < 0.05.
Journal of Investigative Dermatology  , DOI: ( /j x)
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7. Figure 6
ProMMP-2 and activated MMP-2 increase progressively during wound healing and are modulated by GHK-Cu. Densitometric analysis of the zymograms demonstrated that proMMP-2 (A, C) and activated MMP-2 (B, D) increased progressively in both wound fluid (A, B) and wound tissue (C, D) during the first 18 d of wound healing. GHK-Cu injections significantly increased both forms of the enzyme at days 18 and 22. Results are means of three experiments ± 1 SEM. •, controls; ○, GHK-Cu-injected rats. *Significantly different from the corresponding controls, p < 0.05; **p < 0.01
Journal of Investigative Dermatology  , DOI: ( /j x)
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8. Figure 7
GHK-Cu has no direct effect on MMP-2 activity or proMMP-2 activation. Wound fluid from a control chamber was preincubated for 18 h in the presence of increasing concentrations of GHK-Cu. MMP-2 activity was then studied by zymography as described in the text. No effect of GHK-Cu on MMP-2 activity or proMMP-2 activation was observed.
Journal of Investigative Dermatology  , DOI: ( /j x)
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9. Figure 8
GSHK, a peptide of a size similar to GHK is devoid of effect on proMMP-2 expression or activation. Chambers were injected twice a week with either saline (lane 1, control), 2.0 mg GHK-Cu (lane 2) or GSHK at the same molar concentration (lane 3). Chambers were collected on day 18 and the wound fluid analyzed by gelatin zymography. Ten micrograms of protein were applied on each lane.
Journal of Investigative Dermatology  , DOI: ( /j x)
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10. Figure 9
ProMMP-2 and activated MMP-2 proteins are increased in chambers injected with GHK-Cu. Ten micrograms of tissue proteins extracted from six different wound chambers (three controls and three GHK-Cu-injected) were collected 18 d after implantation. They were separated by SDS–PAGE and analyzed by western blotting, using a specific anti-MMP-2 antibody. Lanes 1–3, control rats; lanes 5–7, GHK-Cu injected rats.
Journal of Investigative Dermatology  , DOI: ( /j x)
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11. Figure 10
MMP-2 gene expression increases in the wound tissue from day 3 to day 22. (A) Northern-blot analysis of MMP-2 and glyceraldehyde-3-phosphate-dehydrogenase mRNAs in the wound chambers injected with 2.0 mg GHK(+) or with saline (controls, –) twice a week from day 0 to day 21. The day of sample collection for analysis is indicated. (B) Ratio of MMP-2 to glyceraldehyde-3-phosphate-dehydrogenase mRNAs in wound chambers injected or not (controls, ▪) with GHK-Cu. Results are means of three determinations ± 1 SEM.
Journal of Investigative Dermatology  , DOI: ( /j x)
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12. Figure 10
MMP-2 gene expression increases in the wound tissue from day 3 to day 22. (A) Northern-blot analysis of MMP-2 and glyceraldehyde-3-phosphate-dehydrogenase mRNAs in the wound chambers injected with 2.0 mg GHK(+) or with saline (controls, –) twice a week from day 0 to day 21. The day of sample collection for analysis is indicated. (B) Ratio of MMP-2 to glyceraldehyde-3-phosphate-dehydrogenase mRNAs in wound chambers injected or not (controls, ▪) with GHK-Cu. Results are means of three determinations ± 1 SEM.
Journal of Investigative Dermatology  , DOI: ( /j x)
Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions