1. Re-Epithelialization of Pathological Cutaneous Wounds Is Improved by Local Mineralocorticoid Receptor Antagonism 
Van Tuan Nguyen, Nicolette Farman, Eve Maubec, Dany Nassar, Dorinne Desposito, Ludovic Waeckel, Sélim Aractingi, Frederic Jaisser 
Journal of Investigative Dermatology 
Volume 136, Issue 10, Pages (October 2016)
DOI: /j.jid
Copyright © 2016 The Authors Terms and Conditions

2. Figure 1
MR blockade restores clobetasol-induced impaired wound closure in mouse skin. Quantitative RT-PCR analysis of MR (a), GR (b), and HSD2 (c) mRNA expression in the wound or normal skin in clobetasol (Clo)-treated mice, relative to control (CT). Photographs (d) and quantification of the wound area (e) from mice pretreated 10 days with Clo or PBS, and treated with canrenoate (Canre) or PBS after wounding. (f) Wound sections at day 5 after wounding labeled by an anti-K14 antibody (red) and DAPI (blue). Arrowheads: edges of initial wound and limits of neoepidermis. Quantification of the length of the neoepidermis (g) and the size of the residual wound (h). Anti-Ki67 (green) staining (i) and quantification (j) of Ki67-positive cells in the neoepidermis (Epi) and underlying dermis (Der); dotted lines: dermoepidermis junction. Data are mean values ± SEM. (a–c, g–h, j) Mann-Whitney test; (e) one-way ANOVA followed by the Newman-Keuls multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. Scale bar = 100 μm (f and i). ANOVA, analysis of variance; GR, glucocorticoid receptor; HSD, hydroxysteroid dehydrogenase; K14, keratin-14; MR, mineralocorticoid receptor; PBS, phosphate-buffered saline; RT-PCR, real-time PCR; SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
MR blockade restores impaired keratinocyte outgrowth of clobetasol-treated human skin explants in culture. Human skin explants were collected after 6 days of organotypic culture with/without drugs (CT: control, Clo: clobetasol, and three different MR antagonists, Spiro: spironolactone; Canre: canrenoate; Eple: eplerenone). (a) Photographs of explant sections after H&E staining, showing the neoepidermal tongues growing at the edges of the explants (dotted lines). (b, c) Measurement of the neoepidermal tongues, expressed as length (b) or surface (c). (d) Photographs of Ki67 (green)-labeled explant sections showing proliferating cells; dotted lines indicate the neoepidermal tongues. (e) Quantification of Ki67-positive proliferating keratinocytes, expressed as percentage of total epidermal cells. Data are mean values ± SEM. One-way ANOVA followed by the Newman-Keuls multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001, skin samples from eight subjects. (a, d) Scale bars = 100 μm. ANOVA, analysis of variance; H&E, hematoxylin and eosin; MR, mineralocorticoid receptor; SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
The epithelial sodium channel (ENaC) is involved in wound re-epithelialization. (a–c) Human skin explants in culture: the ENaC blocker phenamil (Phena) improves the clobetasol-induced impaired growth of epidermal tongues, as illustrated on photographs of explant sections stained with H&E (a), and quantified as the length (b) or surface (c) of neoepidermal tongues. (d) Photographs of Ki67 (green)-labeled explant sections showing proliferating cells. (e) Quantification of Ki67-positive proliferating keratinocytes, expressed as percentage of total epidermal cells. Data are mean values ± SEM. One-way ANOVA followed by the Newman-Keuls multiple comparison test. *P < 0.05; **P < 0.01; ***P < Skin samples from eight subjects. Dotted lines in (a) and (d) indicate the neoepidermal tongues. Scale bars = 100 μm. ANOVA, analysis of variance; H&E, hematoxylin and eosin; SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
MR blockade improves the delayed wound closure in type I diabetic mice. Type I diabetes was induced by streptozotocin (STZ). Quantitative RT-PCR analysis of MR (a), GR (b), and HSD2 (c) mRNA expression in the wound or normal skin in STZ, relative to control (CT) mice. Photographs (d) and quantification (e) of the wound area expressed as percentage of wound area at day 0. (f) Wound sections at day 5 after wounding labeled by an anti-K14 antibody (red) and DAPI (blue). Arrowheads: edges of the initial wound and limits of neoepidermis. Quantification of neoepidermis length (g). Anti-Ki67 (green) staining (h) and quantification (i) of Ki67-positive cells in the neoepidermis (Epi) and the underlying dermis (Der); dotted lines: dermoepidermis junction. Data are mean values ± SEM. (a–c, g, i) Mann-Whitney test; (e) one-way ANOVA followed by the Newman-Keuls multiple comparison test. *P < 0.05; **P < 0.01; ***P < 0.001; ns: not significant. Scale bar = 100 μm (f, h). ANOVA, analysis of variance; GR, glucocorticoid receptor; HSD, hydroxysteroid dehydrogenase; K14, keratin-14; MR, mineralocorticoid receptor; RT-PCR, real-time PCR; SEM, standard error of the mean.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

6. Figure 5
Wound closure in healthy volunteers from the SPIREPI clinical study. After 1-month daily application of clobetasol (Clo) alone or associated with spironolactone (Spiro), skin biopsies (3-mm punch) were made, and wounds were photographed using a dermatoscope after biopsy. Photographs of wounds at day 3 (a) and day 7 (c). Surface of the wound measured at day 3 (b) and day 7 (d) (Image J): the lines show the wound surface in each of the four zones (placebo, spironolactone, clobetasol, or both), for each of the 23 subjects included in the SPIREPI trial. Analysis of variance for repeated measures, followed by the Newman-Keuls multiple comparison test. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < SPIREPI, Interest of Topical Spironolactone's Administration to Prevent Corticoid-induced Epidermal Atrophy.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions