The role of estrogen deficiency in skin ageing and wound healing

Abstract

The links between hormonal signalling and lifespan have been well documented in a range of model organisms. For example, in C. elegans or D. melanogaster, lifespan can be modulated by ablating germline cells, or manipulating reproductive history or pregnenolone signalling. In mammalian systems, however, hormonal contribution to longevity is less well understood. With increasing age human steroid hormone profiles change substantially, particularly following menopause in women. This article reviews recent links between steroid sex hormones and ageing, with special emphasis on the skin and wound repair. Estrogen, which substantially decreases with advancing age in both males and females, protects against multiple aspects of cellular ageing in rodent models, including oxidative damage, telomere shortening and cellular senescence. Estrogen's effects are particularly pronounced in the skin where cutaneous changes post-menopause are well documented, and can be partially reversed by classical Hormone Replacement Therapy (HRT). Our research shows that while chronological ageing has clear effects on skin wound healing, falling estrogen levels are the principle mediator of these effects. Thus, both HRT and topical estrogen replacement substantially accelerate healing in elderly humans, but are associated with unwanted deleterious effects, particularly cancer promotion. In fact, much current research effort is being invested in exploring the therapeutic potential of estrogen signalling manipulation to reverse age-associated pathology in peripheral tissues. In the case of the skin the differential targeting of estrogen receptors to promote healing in aged subjects is a real therapeutic possibility.

Full text

REVIEW ARTICLE
The role of estrogen deficiency in skin ageing and wound
healing
Elaine Emmerson •Matthew J. Hardman
Received: 26 November 2010 / Accepted: 11 February 2011
ÓSpringer Science+Business Media B.V. 2011
Abstract The links between hormonal signalling
and lifespan have been well documented in a range of
model organisms. For example, in C. elegans or
D. melanogaster, lifespan can be modulated by
ablating germline cells, or manipulating reproductive
history or pregnenolone signalling. In mammalian
systems, however, hormonal contribution to longev-
ity is less well understood. With increasing age
human steroid hormone profiles change substantially,
particularly following menopause in women. This
article reviews recent links between steroid sex
hormones and ageing, with special emphasis on the
skin and wound repair. Estrogen, which substantially
decreases with advancing age in both males and
females, protects against multiple aspects of cellular
ageing in rodent models, including oxidative damage,
telomere shortening and cellular senescence. Estro-
gen’s effects are particularly pronounced in the skin
where cutaneous changes post-menopause are well
documented, and can be partially reversed by clas-
sical Hormone Replacement Therapy (HRT). Our
research shows that while chronological ageing has
clear effects on skin wound healing, falling estrogen
levels are the principle mediator of these effects.
Thus, both HRT and topical estrogen replacement
substantially accelerate healing in elderly humans,
but are associated with unwanted deleterious effects,
particularly cancer promotion. In fact, much current
research effort is being invested in exploring the
therapeutic potential of estrogen signalling manipu-
lation to reverse age-associated pathology in periph-
eral tissues. In the case of the skin the differential
targeting of estrogen receptors to promote healing in
aged subjects is a real therapeutic possibility.
Keywords Estrogen Ageing Skin Wound
healing
Evolutionary conservation
The links between hormones and ageing, principally
identified through the study of model organisms, are
numerous. The most prominent example would be the
correlation between lifespan and insulin signalling,
which is conserved across numerous species. Indeed,
the importance of endocrine signalling in lower
animal species was initially demonstrated for nema-
tode (Caenorhabditis elegans) DAF-2, a homologue
of the mammalian insulin and insulin-like growth
factor (IGF) receptors, mutations in which lengthened
lifespan (Kimura et al. 1997; Kenyon et al. 1993;
Larsen et al. 1995). Mutations in numerous genes in
the insulin and IGF signalling pathway have also
been shown to prolong the lifespan of Drosophila
melanogaster (Tatar et al. 2001). Mice and higher
E. Emmerson M. J. Hardman (&)
Faculty of Life Sciences, The University of Manchester,
A V Hill Building, Oxford Road,
Manchester M13 9PT, UK
e-mail: matthew.j.hardman@manchester.ac.uk
123
Biogerontology
DOI 10.1007/s10522-011-9322-y

vertebrates, unlike worms and flies, have separate
insulin and IGF receptors. Interestingly, mice hetero-
zygous for IGF-1 receptor live *30% longer than
wild-type mice (Holzenberger et al. 2003), while
mice null for the downstream mediators, insulin
receptor substrate (IRS)-1 and -2, also have extended
lifespan (Selman et al. 2008; Taguchi et al. 2007).
Such extensive cross-species conservation confirms
the importance of this pathway in regulating lifespan
(Liang et al. 2003). In fact, decreasing insulin/IGF
signalling remains one of the few interventions that
does robustly extend murine longevity.
Reproductive state and reproductive system sig-
nals can also influence lifespan. For example, germ-
line precursor cell ablation in C. elegans and
D. melanogaster extends lifespan by *60% (Hsin
and Kenyon 1999; Flatt et al. 2008; Arantes-Oliveira
et al. 2002), a process thought to be regulated via
DAF-2 (Hsin and Kenyon 1999), DAF-9 [a homo-
logue of mammalian cytochrome P450] (Gerisch
et al. 2001; Gerisch and Antebi 2004; Jia et al. 2002)
and DAF-12 (Broue et al. 2007), amongst others
(Fig. 1a). The hormone repertoire of model organ-
isms most commonly used for ageing research, such
as C. elegans and D. melanogaster, is limited, yet
they do express hormone precursors present in higher
animals, such as pregnenolone and essential enzymes
necessary for conversion, such as cytochrome P450
(aromatase) (Broue et al. 2007; Motola et al. 2006).
Signalling via such precursors extends lifespan in
C. elegans (Yamawaki et al. 2010; Broue et al.
2007; Mak and Ruvkun 2004) and D. melanogaster
(Yamawaki et al. 2010; Simon et al. 2003). For
example, pregnenolone extends lifespan in nema-
todes, via DAF-9 (Broue et al. 2007) (Fig. 1b).
Surprisingly, the relevance of steroid precursors, such
as pregnenolone, in mammalian longevity is largely
unknown. Interestingly, aged ovariectomised (Ovx)
mice transplanted with the ovaries of young mice
exhibit significantly increased life expectancy
(Mason et al. 2009; Cargill et al. 2003), an effect
potentially mediated through the estrogen receptors
and/or the IGF-1R (Fig. 1c).
Ageing in humans, the menopause and pathology
With increasing age the human endocrine system
undergoes substantial change, particularly with
respect to hormones of adrenal origin. The steroid
precursor dehydroepiandrosterone (DHEA), its sul-
phate, DHEA-S, and the precursor androstendione
decline substantially in both males and females from
age 20 (Labrie et al. 1997). Interestingly, serum
concentrations of pregnenolone (discussed above)
also decrease with age in both males and females
(Havlikova et al. 2002; Labrie et al. 1997). In women
levels of sex steroid estrogens begin to fall from
approximately 35 years of age and follicle stimulat-
ing hormone (FSH) production is increased in an
effort to stimulate ovarian function (Chakravarti et al.
1976). While 17b-estradiol, the most potent form of
estrogen, decreases with age serum estrone concen-
trations remain fairly constant (Labrie et al. 1997).
The most pronounced changes occur when women
enter the menopause, a permanent cessation of
menstruation resulting from the loss of ovarian
follicular activity, which occurs at an average age
of 51 years in the developed world (Stanford et al.
Fig. 1 Association between steroid sex hormones and life-
span. aGermline cell ablation in C. elegans and D. melano-
gaster extends lifespan via DAF-2, -9 and -12. bPregnenolone
signals via DAF-9 and extends lifespan in C. elegans.
cHormone transfer by ovary transplant from young mice to
aged mice extends lifespan, an effect presumably mediated by
estrogen and the ERs or IGF-1R
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123

1987). Post-menopause the majority of circulating
sex steroids actually originate from circulating adr-
enally-derived DHEA (Labrie et al. 2011). In aged
males estrogen levels also decrease, although not as
rapidly as in females. By contrast, testosterone and
dihydrotestosterone (DHT) fluctuate but remain fairly
constant with age in both sexes (Labrie et al. 1997).
Of note, the enzyme 11b-hydroxysteroid hydrogenase
(HSD), which is responsible for the conversion of
cortisone to cortisol, is markedly increased with age
(Vukelic et al. 2011; Tiganescu et al. 2011).
Increasing life expectancy now means that the
average woman in the developed world spends one-
third of her life in the post-menopausal period
(Kligman and Koblenzer 1997). It is widely accepted
that decreased systemic hormones in the post-men-
opausal state is associated with increased risk for a
range of age-associated pathologies (Table 1).
Estrogen deficiency as a general mechanism
of ageing
Arguably the most profound hormonal change in
ageing is the post-menopause reduction in 17b-
estradiol. Moreover, a substantial body of literature
links estrogen decline to the distinct cellular ageing
mechanisms of oxidative damage and cellular
senescence.
Oxidative damage
Free radicals are strongly implicated in the cellular
damage that accompanies ageing and age-associated
disease (Harman 1956; Beckman and Ames 1998),
part of the ‘mitochondrial theory of ageing’ (Miquel
et al. 1980). The link between mitochondria and
longevity comes from the observation that by reduc-
ing mitochondrial peroxidase production lifespan can
be extended (Lopez-Torres et al. 1993; Ku et al.
1993). Pertinent to this review is the observation that
both peroxidase production and mitochondrial DNA
damage are significantly (40–80%) higher in male
rats than in age-matched females, and this is directly
attributed to differences in estrogen levels (Borras
et al. 2003; Pinto and Bartley 1968,1969). Mito-
chondrial glutathione, a biological marker of ageing
and age-associated damage (Hazelton and Lang 1984;
Sastre et al. 2000) is significantly higher in male rats
than females (Vina et al. 2005). Conversely, expres-
sion of 16S ribosomal RNA, a biological marker of
youth (Calleja et al. 1993), is substantially higher in
female rats than males (Borras et al. 2003; Vina et al.
2005). Although, Sanz et al. (2007) report no
difference in oxidative stress, age-related damage or
lifespan between male and female C57Bl6 mice, Ali
et al. (2006) show that females, the reported shorter
lived gender, exhibit enhanced reactive oxygen
species (ROS) production with age.
Table 1 Chronic pathologies
associated with ageing and
menopause
Pathology Age Menopause
Auditory degeneration Weinstein and Ventry (1982) Hederstierna et al. (2010)
Hederstierna et al. (2007)
Cancer Yancik et al. (2001) Trichopoulos et al. (1972)
Heart disease Lindblad et al. (2001) Lokkegaard et al. (2006)
Lye and Donnellan (2000) Jacobsen et al. (1997)
Colditz et al. (1987) Barrett-Connor (1995)
Muscular degeneration Evans (2010) Calmels et al. (1995)
Neurodegeneration Kukull et al. (2002) Simpkins et al. (2005)
Suthers et al. (2003) Morrison et al. (2006)
Optical degeneration Owsley et al. (2007) Siesky et al. (2008)
Carcenac et al. (2009) Evans et al. (1998)
Osteoporosis Gates et al. (2009) Melton et al. (1992)
Simonen and Mikkola (1991)
Skin ageing Lavker (1979) Brincat et al. (1987)
Gilchrest et al. (1983) Sumino et al. (2004)
Urinary incontinence Williams and Pannill (1982) Waetjen et al. (2009)
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Estrogen deficiency post-menopause is also
strongly linked to altered oxidative state, with
estrogen a potent direct antioxidant and indirect
inducer of antioxidant enzymes. In ovariectomised
(Ovx) female rats oxidised glutathione, lipid perox-
idation and mitochondrial DNA damage are signif-
icantly increased, and can be reversed by estrogen
replacement or phytoestrogen treatment (Baeza et al.
2010). In vitro both estradiol and the phytoestrogen
genistein reduce the production of hydrogen peroxide
in MCF-7 cells (Borras et al. 2005,2006). Addition-
ally, keratinocytes isolated from aged female rats
exhibit increased oxidative stress (lipoperoxides) and
apoptosis (caspases 3 and 8), which can be reversed
by estrogen treatment (Tresguerres et al. 2008).
Finally, it should be noted that while gender-specific
effects are common across many species, including
man (Gurwitz 2005), marsupials (Humphries and
Stevens 2001) and non-human primates (Herndon
et al. 1999) this is not the case in all animal species.
Some species lack gender difference in lifespan (Sanz
et al. 2007; Wich et al. 2004) while in a limited
number of species males actually live longer (Asdell
and Joshi 1976; McCulloch and Gems 2003).
Senescence
Significant telomere shortening is associated with
replicative cell senescence and tissue deterioration
(Allsopp et al. 1992; Harley 1991), and prevented by
the enzyme telomerase (Morin 1989). The expression
of the human telomerase reverse transcriptase
(hTERT) gene and telomerase activity (TA) is
upregulated in hepatocytes in vitro in response to
estradiol, leading to maintained telomere length (Sato
et al. 2004). Similarly, estrogen dose-dependently
prevents cellular senescence and increases telomerase
activity in endothelial progenitor cells (Imanishi et al.
2005b), vascular smooth muscle cells (Ling et al.
2006) and leukocytes (Aviv et al. 2006) in vitro.In
endothelial progenitor cells estrogen prevents angio-
tensin-II-mediated oxidative stress and senescence
(Imanishi et al. 2005a). Estrogen deficiency signifi-
cantly inhibits TERT expression and telomerase
activity in the mouse adrenal gland, an effect
reversed by estrogen replacement (Bayne et al.
2008). It has thus been speculated that gender specific
differences in longevity are, at least in part, due to
hormonal regulation of telomere function (Aviv et al.
2005; Stindl 2004). One method of detecting senes-
cent cells in vitro is a modified beta-galactosidase (b-
gal) assay (Dimri et al. 1995). Interestingly, the soy-
derived phytoestrogen genistein suppresses UVB-
induced expression of b-gal in primary human dermal
fibroblasts (Wang et al. 2010), while soybean extract
protects against cellular senescence in HaCaT cells
(Chiu et al. 2009).
Skin ageing and estrogens
With increasing age a combination of intrinsic and
extrinsic factors (primarily UV exposure) lead to skin
deterioration. Aged skin has altered structure and
reduced function, particularly loss of elasticity,
wrinkling, thinning and fragility. While there are
differences in the etiology of intrinsic and extrinsic
ageing (Tsoureli-Nikita et al. 2006) the majority of
resultant changes are similar (Tsoureli-Nikita et al.
2006; Pillai et al. 2005; Ashcroft et al. 1997e; Varani
et al. 2001; Lavker 1979). Specifically, intrinsic
ageing involves thinning of the epidermis and dermis,
reduced epidermal proliferation and turnover and
reduced vascularity (Gilchrest et al. 1982b; Lavker
1979). Collagen production is reduced (Shuster et al.
1975) and distribution is altered (Richard et al. 1993),
while the production of matrix degrading enzymes
(MMPs) is increased with intrinsic ageing (Ashcroft
et al. 1997e). Extrinsic ageing or photoageing
involves dermal elastosis (Mitchell 1967; Braverman
and Fonferko 1982), reduced Langerhans cell num-
bers, altered melanocyte distribution (Thiers et al.
1984; Gilchrest et al. 1982a) and increased MMP
activity (Pillai et al. 2005). With advancing age
reduced sebaceous gland secretion leads to skin
xerosis, an event that involves corticotropin-releasing
hormone (CRH) and coincides with the onset of
menopause in women (Pochi et al. 1979; Zouboulis
et al. 2002).
The effects of ageing can be readily observed in
skin cells in vitro, where dermal fibroblasts and
epidermal keratinocytes from aged donors have
reduced proliferative capacity and display premature
senescence (Stanulis-Praeger and Gilchrest 1989;
Gilchrest 1983; Schneider and Mitsui 1976; Mets
et al. 1983). In ageing human skin senescent cells
accumulate through a combination of growth arrest
and increased resistance to apoptosis (Dimri et al.
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1994; Wang 1995). In the principal skin cell types
(keratinocytes/fibroblasts) telomeres shorten an aver-
age of 9 and 11 base pairs per year, respectively,
throughout life (Sugimoto et al. 2006; Krunic
et al. 2009). Interestingly, telomere shortening is
reportedly unchanged in sun-exposed (extrinsically
aged) versus sun-protected (intrinsically aged) skin
(Sugimoto et al. 2006). Instead, large deletions of the
mitochondrial genome are believed to be involved in
UV-induced skin photoageing (Schroeder et al.
2008). In vitro, repetitive exposure of human dermal
fibroblasts to UVA has been shown to induce
deletions of up to 5000 base pairs, causing a partial
loss of the mitochondrial genome (Berneburg et al.
1999). Moreover, in a model where mtDNA deletions
were induced without irradiation, gene expression
mirrored that observed in photoaged skin (Schroeder
et al. 2008).
While laboratory animals provide excellent trac-
table models, some aspects of skin ageing, e.g.,
changes in epidermal thickness, are contentious and
appear species and strain dependent. The majority of
mouse studies find that with age epidermal thickness
is reduced (Iversen and Schjoelberg 1984; Haratake
et al. 1997; Bhattacharyya and Thomas 2004;
Argyris 1983) and epidermal keratinocyte prolifer-
ation is decreased (Cameron 1972). However, other
studies have reported increased epidermal thickness
with age (Hill 1988). Epidermal thickness in aged
rats has been reported to increase (Bhattacharyya
et al. 2005; Thomas 2005), decrease (Morris et al.
1990) or remain unchanged (Giangreco et al. 2008;
Monteiro-Riviere et al. 1991). Interestingly, Ishib-
ashi rats (progeny of Wistar and wild rats) report-
edly undergo qualitatively similar skin changes to
humans. From 12 weeks of age wrinkling of the
skin occurs, consistent with a reduction in elastin
(Sakuraoka et al. 1996). Calorie restriction (CR),
which extends lifespan in numerous model organ-
isms (S. cerevisiae,C. elegans,D. melanogaster)
(Lin et al. 2002; Klass 1977; Hosono et al. 1989;
Mair et al. 2003; Pletcher et al. 2002) and higher
mammals, such as mice and non-human primates
(Lane et al. 1995; McCay et al. 1935; Rezzi et al.
2009; Ramsey et al. 2000), prevents age-related skin
changes when compared to age-matched ad libitum
controls (Thomas 2005; Bhattacharyya et al. 2005).
Unfortunately, neither of these studies specifies the
sex of the experimental animals.
Historic studies provide the first evidence of
estrogen’s cutaneous effects: topically applied follic-
ular hormone, now known to be estrogen, was found
to locally improve acne and eczema (Loeser 1937). It
has also long been known that the symptoms of skin
disorders such as psoriasis improve during preg-
nancy, an observation now directly attributed to
increased circulating estrogen (Dunna and Finlay
1989; Boyd et al. 1996) Moreover, the oral contra-
ceptive pill is often prescribed to treat severe acne
(Arowojolu et al. 2009). During the menopause, skin
undergoes major changes which include reduced
epidermal and dermal thickness, a decrease in
collagen content (Brincat et al. 1987), reduced
elasticity (Sumino et al. 2004), dryness and fragility
(Brincat et al. 1985). Crucially, the majority of these
changes can be reversed by either topical or systemic
hormone replacement therapy (HRT) (Table 2). Not
surprisingly, topical estrogen treatment has little
effect on young skin (Goldzieher 1949). The effects
of estrogen are predominantly mediated through the
estrogen receptors (see ‘‘Estrogen receptors and
SERMs’’ section). Estrogen also protects against
photoageing, with mortality rates from both mela-
noma (Miller and Mac Neil 1997) and non-melanoma
(Weinstock 1994) skin cancer lower in women than
men. In experimental animal studies estrogen depri-
vation enhances sensitivity to UV skin damage and
accelerates photoageing, measured as wrinkling, a
loss of elasticity and damage to elastic fibres
(Tsukahara et al. 2001; Tsukahara et al. 2004), while
estrogen treatment increases skin collagen content in
rats and guinea pigs (Smith and Allison 1966;
Henneman 1968), increases hyaluronic acid synthesis
(Sobel et al. 1965) and promotes epidermal thicken-
ing in mice (Bullough 1947). A potential link
between ageing and estrogen involves IGF-1. Estra-
diol is known to be able to signal through the IGF-1R
in the skin, in a non-genomic manner, strongly
implicating it’s involvement in the regulation of
cutaneous ageing (Makrantonaki et al. 2008;
Surazynski et al. 2003).
In reality, the skin is not only an estrogen target,
but also a major synthetic organ with the capacity to
release and produce a wide range of hormones,
including estrogens. Skin cells are able to synthesise
locally acting estrogens from the precursors choles-
terol and DHEA (Fig. 2) (Simpson et al. 1997; Bulun
et al. 1999; Chen et al. 2002; Chen et al. 1998;
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Thiboutot et al. 1998; Hughes et al. 1997; Sawaya
and Penneys 1992; Dumont et al. 1992; Eicheler et al.
1995; Thiboutot et al. 2000). Additionally, local
production of 11b-HSD by epidermal keratinocytes,
dermal fibroblasts and hair follicles (Tiganescu et al.
2011) increases cortisol conversion and has been
speculated to control the inflammatory response
(Vukelic et al. 2011). In aged individuals peripheral
hormone production and local hormone signalling
are likely to be particularly important, in light
of the considerable reduction in systemic hormone
production (Longcope 1971). Surprisingly, the effects
of ageing on peripheral hormone synthesis/conver-
sion are virtually unknown.
Wound repair with age
Cutaneous wound healing is a complex tightly
orchestrated response to injury, carefully regulated
at temporal and spatial levels (reviewed in Shaw and
Martin 2009). In young individuals the innate
Table 2 Cutaneous changes
with post-menopausal
estrogen replacement
Cutaneous changes Reference(s)
Increased epidermal thickness Punnonen (1971), Brincat (2000), Fuchs et al. (2003)
Increased dermal thickness Son et al. (2005), Shuster et al. (1975), Brincat et al. (1985)
Increased collagen content Castelo-Branco et al. (1992), Savvas et al. (1993), Shuster
et al. (1975), Varila et al. (1995)
Increased elastin content Punnonen et al. (1987)
Increased skin moisture content Pierard-Franchimont et al. (1995), Schmidt et al. (1994)
Improved external appearance Schmidt et al. (1996), Schmidt et al. (1994), Brincat et al.
(1985), Creidi et al. (1994)
Fewer wrinkles Aertgeerts (1974)
Fig. 2 Skin contains all the components for peripheral
steroid hormone synthesis. The precursors cholesterol and
dehydroepiandrosterone (DHEA) can be converted to steroid
hormones by aromatase (the product of the CYP19 gene), 3b-
hydroxysteroid dehydrogenase (3b-HSD), 17b-hydroxysteroid
dehydrogenase (17b-HSD) and 5a-reductase. 17a-estradiol has
been shown to influence the conversion of testosterone to 17b-
estradiol and androstenedione. Double boxes represent func-
tionally important non-steroid hormone synthesis
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inflammatory response is first initiated, followed by a
widespread proliferative phase, involving fibroblasts,
keratinocytes and endothelial cells. Keratinocyte
migration restores the skin’s barrier, fibroblast-med-
iated contraction aids wound closure, while matrix
remodelling leads to a mature scar. With the passage
of time skin becomes fragile and prone to trauma,
while cellular ageing leads to aberrant healing.
Platelet function (Boldt et al. 2001; Fukaya et al.
2000) and platelet-derived growth factor (PDGF)
expression (Ashcroft et al. 1997d) is altered with age.
The inflammatory response becomes disrupted, with
excessive neutrophil influx, altered endothelial cell
adhesion, prolonged macrophage recruitment and
delayed resolution (Ashcroft et al. 1998). This leads
to over-production of matrix degrading enzymes,
classically elastase and MMPs (Ashcroft et al. 1997e;
Ashcroft et al. 1997f; Herrick et al. 1997), and under-
expression of tissue inhibitors of metalloproteinases
(TIMPs) (Ashcroft et al. 1997b). In vivo, re-epithel-
ialisation is delayed in aged humans and murine
models (Ashcroft et al. 1997a). Corroborative in vitro
studies show that keratinocytes from young donors
proliferate at a faster rate (Stanulis-Praeger and
Gilchrest 1986) and exhibit less sensitivity to epider-
mal growth factor (EGF) and keratinocyte growth
factor (KGF) (Gilchrest 1983) than those from old
aged donors. Angiogenesis is delayed in aged humans
and mice (Ashcroft et al. 1997c; Sadoun and Reed
2003) and collagen deposition is reduced (Lenhardt
et al. 2000; Ashcroft et al. 1997c). Ultimately, in
elderly subjects scar strength is reduced (Lindstedt
and Sandblom 1975; Sandblom et al. 1953; Mendoza
et al. 1970), however, scar quality is improved
(Ashcroft et al. 1999).
Repair, ageing and hormones
While chronological age is a clear risk factor for poor
healing our recent studies suggest that estrogen
deprivation is the major factor controlling delayed
healing in elderly humans. In our recent microarray
study 78% of genes differentially expressed between
wounds from young and elderly men were estrogen-
regulated, while only 3% were age-associated,
strongly implicating reduced estrogen, and not known
gerontogenes, as the primary regulator of delayed
healing in aged subjects (Hardman and Ashcroft
2008). In post-menopausal women HRT improves
healing (Ashcroft et al. 1997a), while topical estrogen
treatment improves healing in elderly subjects of
either gender (Ashcroft et al. 1999). Estrogen’s
cellular effects include dampening excessive neutro-
phil recruitment, preventing disproportionate elastase
production, and increasing fibronectin and collagen
deposition. Crucially, two independent studies reveal
that HRT protects post-menopausal women from
developing venous leg ulcers or pressure ulcers
(Margolis et al. 2002; Berard et al. 2001).
Animal models have provided key insight into
estrogen’s role in wound healing (summarised in
Fig. 3). Estrogen replacement accelerates cutaneous
healing in Ovx female mice and rats (Ashcroft et al.
Fig. 3 Mechanism of
estrogen activity in older
people. Estrogen aids
healing through effects on
fibroblasts, keratinocytes
and inflammatory cells
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1997a; Ashcroft et al. 2003; Emmerson et al. 2010;
Hardman et al. 2008) and male rats (Rajabi and
Rajabi 2007). Systemic treatment with the sex steroid
precursor DHEA accelerates wound healing in young
Ovx female mice and old male mice, a result
attributed to the local conversion of DHEA to
estrogen (Mills et al. 2005). Of interest, androgens
are detrimental to healing. The testosterone metabo-
lite DHT retards repair (Gilliver et al. 2009), while
castration or androgen receptor blockade improves
healing in rodents (Gilliver et al. 2003; Gilliver et al.
2006; Ashcroft and Mills 2002).
At the cellular level estrogen down-regulates
neutrophil-expressed L-selectin, preventing the
excessive neutrophil accumulation and neutrophil-
derived elastase production characteristic of aged
healing (Ashcroft et al. 1999), and improves neutro-
phil phagocytic ability (Magnusson and Einarsson
1990). Estrogen dampens expression of numerous
pro-inflammatory cytokines, including MIF, TNFa,
MCP-1, IL-1band IL-6 (Kovacs et al. 1996; Hu et al.
1988). The reduction in Macrophage Migration
Inhibitory Factor (MIF), in particular, plays a major
role in the beneficial effects of estrogen on wound
repair (Ashcroft et al. 2003; Hardman et al. 2005;
Emmerson et al. 2009). Of interest, plasma MIF
levels, which inversely correlate with systemic
estrogen (Aloisi et al. 2005), are increased post-
menopause and fall following HRT (Hardman et al.
2005). Estrogen is also a keratinocyte mitogen
(Verdier-Sevrain et al. 2004), promoting migration
across an artificial scratch in vitro (Emmerson et al.
2009; Campbell et al. 2010) and wound re-epithel-
ialisation in Ovx mice (Emmerson et al. 2010;
Hardman et al. 2008). Moreover, impaired wound
re-epithelialisation post-menopause can be reversed
to pre-menopause levels by just 3 months of HRT
(Ashcroft et al. 1997a). Estrogen directly stimulates
dermal fibroblast migration in vitro (Emmerson et al.
2009; Campbell et al. 2010) and indirectly via
stimulating macrophage platelet derived growth fac-
tor (PDGF), a key fibroblast mitogen and stimulator
of wound contraction (Battegay et al. 1994). The role
of estradiol in stimulating angiogenesis is somewhat
contentious. PDGF directly stimulates angiogenesis,
while estradiol increases endothelial cell capillary-
like structure formation upon reconstituted basement
membrane (Morales et al. 1995). However, conflict-
ing in vitro studies report either no change or
decreased angiogenesis following estrogen treatment
(Nyman 1971; Lundgren 1973).
Estrogen receptors and SERMs
Estrogen signals via two nuclear hormone receptors,
ERaand ERb.ERa, the first receptor to be identified
and cloned (Walter et al. 1985), predominates in
reproductive tissues and is strongly associated with
cancer (Zou and Ing 1998; Kuiper et al. 1997; Ali and
Coombes 2000). The second receptor, ERb, identified
over a decade later (Kuiper et al. 1996; Ogawa et al.
1998), is more highly expressed in peripheral, non-
reproductive tissues (Kuiper et al. 1997; Onoe et al.
1997; Brandenberger et al. 1997; Couse et al. 1997).
Both receptors are reportedly expressed in human
facial skin (Miller and Mac Neil 1997), scalp (Thorn-
ton et al. 2003a,b; Mosselman et al. 1996) and upper
arm skin (Reed et al. 2005). Several studies suggest that
ERbpredominates in keratinocytes of human scalp
skin (Mosselman et al. 1996; Thornton et al. 2003a,b)
while others identify both receptors in neonatal
foreskin-derived keratinocytes (Verdier-Sevrain et al.
2004). In mouse skin both receptors are widely
expressed (Campbell et al. 2010; Cho et al. 2008).
Our recent data indicate that delayed healing in
Ovx female mice can be reversed by stimulating
signalling through ERbalone (using the ERb-specific
agonist DiarylPropioNitrile [DPN]) (Campbell et al.
2010). Conversely, signalling through ERaalone
(using the ERaagonist Propyl Pyrazole Triol [PPT])
entirely fails to improve healing in Ovx mice. To
support this finding, we find that estrogen replace-
ment in Ovx mice lacking functional ERb(ERb-/-)
actually further delays healing beyond the Ovx wild-
type phenotype. Moreover, epidermal specific ERb
null mice (K14-cre/ERb
L2/L2
) phenocopy ERb-/-
mice, i.e., estrogen replacement again impairs heal-
ing. This would suggest that the beneficial effects of
estrogen on cutaneous repair are predominantly
mediated via epidermal ERb(Campbell et al.
2010). Of note, the beneficial effects of 17b-estradiol
on outcome in a skin flap necrosis model is reportedly
mediated via ERa(Toutain et al. 2009) while Ovx
rats treated with PPT exhibit reduced wound tensile
strength (Gal et al. 2010). A crucial link between our
mouse data and aged human healing is provided by
the observation that polymorphisms in the human
Biogerontology
123

ERbgene are significantly associated with venous
ulceration in the Caucasian population (Ashworth
et al. 2005,2008).
Most pathological states in the elderly involve a
single ER isoform, for example in cancers of the
reproductive system ERapredominates (Herynk and
Fuqua 2004; Yang et al. 2008; Cai et al. 2003), while
in colon cancers ERbpredominates (Arai et al. 2000;
Fiorelli et al. 1999; Foley et al. 2000; Qiu et al. 2002).
The clear differential roles of the two estrogen
receptors in cutaneous repair (Campbell et al. 2010;
Gal et al. 2010; Toutain et al. 2009) suggest that
pharmacological manipulation may be a viable ther-
apeutic option. Compounds termed Selective Estro-
gen Receptor Modulator (SERMs) are being
developed to treat pathologies such as osteoporosis
and breast cancer by exploiting natural estrogen
signalling to confer tissue specific estrogenic or anti-
estrogenic effects.
SERMs have been developed to provide estrogen-
like beneficial effects, to treat disorders such as
osteoporosis, an approach that should bypass
systemic estrogen risks, including breast cancer
(Matsumoto 2006). Arguably, the best characterised
and commonly used SERMs are tamoxifen and
raloxifene. Both are considered ER antagonists in
the breast, but have the potential to act as ER agonists
in other tissues (Frasor et al. 2004). The dietary
phytoestrogen genistein, is also becoming increas-
ingly popular for the treatment of post-menopausal
pathology (Atteritano et al. 2008; D’Anna et al.
2007). Despite use of SERMs in age- and menopause-
associated pathologies (reviewed in Pickar et al.
2010) knowledge of SERM activity in the skin is
severely lacking. In vitro both tamoxifen and raloxif-
ene stimulate fibroblast proliferation, but fail to
promote fibroblast migration (Stevenson et al.
2009). Preliminary studies in post-menopausal skin
indicate increased elasticity and collagen content
following raloxifene treatment (Sumino et al. 2009)
and improved dermal vascularisation and increased
epidermal thickness following genistein treatment
(Moraes et al. 2009). Topical tamoxifen reportedly
improves the appearance of keloid scars in acute
burns patients, through dampening of fibroblast
proliferation and collagen synthesis (Mousavi et al.
2010; Gragnani et al. 2009). Our recent studies
indicate that the SERMs tamoxifen, raloxifene and
genistein all substantially benefit healing in the
estrogen-deprived Ovx mouse, promoting re-epithel-
ialisation and contraction, and reducing inflamma-
tion, an effect that is most likely mediated via ERb
(Emmerson et al. 2010; Hardman et al. 2008), while a
separate study provides evidence that genistein
accelerates murine healing by modulating TGFb1
(Marini et al. 2010). Tamoxifen, raloxifene and
genistein are anti-inflammatory in other pathologies,
including systemic lupus erythematosus (SLE)
(Sthoeger et al. 2003), stroke (Tian et al. 2009),
UV-associated cutaneous damage (Brand and
Jendrzejewski 2008; Shyong et al. 2002; Widyarini
et al. 2001), colitis (Seibel et al. 2009), ileitis
(Sadowska-Krowicka et al. 1998) and multiple scle-
rosis (MS) (De Paula et al. 2008). An important and
on-going goal of our research is to evaluate cutaneous
healing in post-menopausal women prescribed topical
SERM treatment.
Conclusions and further perspectives
Despite links between estrogen and ageing being
suggested many years ago, only over the last decade
has estrogen emerged as a key determinant of ageing
in peripheral non-reproductive tissues, particularly
bone, skin and brain. Indeed, the endocrine theory of
ageing, underpinned by germline cell ablation exper-
iments in model organisms, states that chronological
changes in hormones levels accelerate the cellular
effects of ageing. In skin particularly, it is only over
the last few years that we have begun to understand
the relative contributions of hormones and ageing to
pathological healing. Indeed, although our knowledge
of estrogen’s beneficial effects on wound repair has
greatly expanded over recent years there is still much
that is not understood. The prospect of manipulating
estrogen signalling to alleviate poor healing in the
elderly is an exciting one, but we have yet to fully
understand how such ER-mediated signalling
changes with normal ageing and how this impacts
upon cutaneous pathology post-menopause. Impor-
tantly, how do the protective effects of estrogen on
cellular ageing (i.e., prevention of telomere shorten-
ing and oxidative stress) directly contribute to
improved healing? A clearer detailed mechanistic
understanding will be of relevance to a range of
peripheral estrogen-target tissues and aid the
Biogerontology
123

development of targeted hormone-based therapies, to
promote cutaneous repair in the elderly.
Acknowledgments Dr Emmerson is the David Hammond
Charitable Trust Research Associate (The Healing Foundation)
and recipient of the BSRA 2010 Korenchevsky award. Dr
Hardman is the Edmund de Rothschild Senior Fellow in
Ageing Research (Research Into Ageing; Age UK).
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