1. The Sex Steroid Precursor DHEA Accelerates Cutaneous Wound Healing Via the Estrogen Receptors 
Stuart J. Mills, Jason J. Ashworth, Stephen C. Gilliver, Matthew J. Hardman, Gillian S. Ashcroft 
Journal of Investigative Dermatology 
Volume 125, Issue 5, Pages (November 2005)
DOI: /j X x
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Dehydroepiandrosterone (DHEA) accelerates impaired wound healing in estrogen-depleted mice. (a) Systemic levels of DHEA were reduced in human subjects with, or who had suffered from, chronic venous ulceration (details in Materials and Methods). Results shown are mean±SEM. *p<0.05, **p<0.01. Spearman's coefficient for male data=0.232, and female= (b) Administration of systemic DHEA over 3 d significantly reduced wound areas in ovariectomized (OVX) mice, n=6 per group. Results shown are mean±SEM. *p<0.05, **p<0.01. Hematoxylin and eosin (H&E) images represent day 3 wound sections (arrows denote the wound edge). Vehicle, OVX with vehicle treatment. OVX, OVX unmanipulated. Scale bars=0.25 mM. (c) DHEA treatment over 3 d reduced the numbers of macrophages (Mac-3) and neutrophils (Ly-6G) within the wound as illustrated by immunostaining (scale bars=0.025 mM) and cell counts (graph). Results shown are mean±SEM. *p<0.05 **p<0.01 (compared to vehicle control). Collagen levels were increased in wound tissue following DHEA treatment (arrows indicate positive staining of matrix collagen, scale bars=0.25 mM).
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Conversion of dehydroepiandrosterone (DHEA) to metabolites by aromatase is necessary for its effects on wound healing. (a) Panels show representative H&E-stained sections of day 3 wounds from ovariectomized (OVX) mice treated with vehicle, three daily treatments with 10−7 M s/c DHEA, or DHEA co-administered with arimidex. Scale bars=0.25 mM. Graph illustrates cross-sectional wound areas as determined in Methods. *p<0.05 (b) Panels shown Mac-3 staining. The graph shows that the reduction in wound macrophage numbers (*p<0.05) resulting from DHEA treatment is reversed when arimidex is co-administered. (c) Panels show Ly-6G staining. The graph shows that the reduction in macrophage number (*p<0.05) resulting from DHEA treatment is reversed when arimidex is co-administered. All graphical results shown are mean±SEM, n=6 per group.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Administration of systemic dehydroepiandrosterone (DHEA) reduced wound tissue levels of pro-inflammatory cytokines. Cell counts (graphs) for (a) macrophage migration inhibitory factor (MIF), (b) tumor necrosis factor-alpha (TNF-α) and (c) interleukin 6 (IL-6) were significantly reduced in day 3 wounds where mice had been treated with DHEA (three doses), and arimidex reversed the effects of DHEA in all cases. Results shown are mean±SEM. *p<0.05, **p<0.01, n=6 per group. Panels show representative immunohistochemical staining of cells expressing (a) MIF, (b) TNF-α and (c) IL-6, illustrating reduced numbers of positively-staining cells (arrows) following DHEA treatment, and was confirmed by western blotting of wound tissue (β-actin acted as loading control). Graphs (on right of panel) represent real time PCR for MIF, TNF-α and IL-6 (all repeated in triplicate). Results shown are mean±SEM. *p<0.05. n=6 per group. Ovariectomized (OVX), OVX vehicle treated.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Dehydroepiandrosterone (DHEA) acts via the estrogen receptor to modulate wound healing. (a) Panels show hematoxylin and eosin (H&E) staining of day 3 wounds (arrows demarcate wound margins). Scale bars=0.25 mM. Graph represents the reduction in wound area following DHEA treatment for 3 d (*p<0.01) and the reversal of these effects by ICI (estrogen receptor (ER) inhibitor). (b) Panels show that the DHEA-mediated reduction in numbers of cells staining positively for Mac-3 is abolished when DHEA is co-administered with ICI. Scale bars=0.025 mM (c) DHEA inhibition of macrophage migration inhibitory factor (MIF), interleukin 6 (IL-6), and tumor necrosis factor-alpha (TNF-α) protein (as determined by ELISA) and (d) mRNA (real-time PCR) was abolished by arimidex (*p<0.05). ICI also reversed the DHEA effects on MIF, but not IL-6 and TNF-α, production. For all graphs, results shown are mean±SEM, all experiments carried out in triplicate, n=6 per group.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Dehydroepiandrosterone (DHEA) regulates MIF through MAP/PI3 kinase pathways in macrophages. (a) Representative ERK1/2 immunoblotting: levels were increased in wound tissue following ovariectomization (OVX). DHEA had no effect on protein levels. (b) DHEA-mediated inhibition of macrophage macrophage migration inhibitory factor (MIF) production was reversed when co-incubated with PD 98,059 and wortmannin when compared to the DHEA group. (c) In the case of tumor necrosis factor-alpha (TNF-α) (and interleukin 6 L-6), data not shown) DHEA inhibited production but PI3/MAP kinase inhibitors had no effect on DHEA responses. For all graphs. (a–d) results shown are mean±SEM, *p<0.01, **p<0.001, all experiments carried out in triplicate, n=6 per group.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Dehydroepiandrosterone (DHEA) accelerates healing in an ageing mouse colony. (a,b) Hematoxylin and eosin (H&E) stained slides (day 7) and graph illustrates wound areas at days 3, 7 and 14 post-wounding. A significant increase in area in the old mice compared to the young at day 7 was observed, and a DHEA-mediated reduction in area in the aged mice at days 3 and 7. Results shown are mean±SEM. *p<0.05, **p<0.01, n=6 per group. (c) Collagen I levels were reduced in the aged mouse wounds at day 7 compared to the young, and a DHEA-mediated increase in the aged. Arrows represent positive staining for collagen. Scale bar=0.25 mM.
Journal of Investigative Dermatology  , DOI: ( /j X x)
Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions