Insulin-like growth factor-1 promotes wound healing in estrogen-deprived mice: new insights into cutaneous IGF-1R/ERα cross talk.

Although it is understood that endogenous IGF-1 is involved in the wound repair process, the effects of exogenous IGF-1 administration on wound repair remain largely unclear. In addition, the signaling links between IGF-1 receptor (IGF-1R) and estrogen receptors (ERs), which have been elucidated in other systems, have yet to be explored in the context of skin repair. In this study, we show that locally administered IGF-1 promotes wound repair in an estrogen-deprived animal model, the ovariectomized (Ovx) mouse, principally by dampening the local inflammatory response and promoting re-epithelialization. Using specific IGF-1R and ER antagonists in vivo, we reveal that IGF-1-mediated effects on re-epithelialization are directly mediated by IGF-1R. By contrast, the anti-inflammatory effects of IGF-1 are predominantly via the ERs, in particular ERα. Crucially, in ERα-null mice, IGF-1 fails to promote healing, and local inflammation is increased. Our findings illustrate the complex interactions between IGF-1 and estrogen in skin. The fact that IGF-1 may compensate for estrogen deficiency in wound repair, and potentially other contexts, is an important consideration for the treatment of postmenopausal pathology.

Tumor necrosis factor-alpha (TNF-α) is a therapeutic target for impaired cutaneous wound healing.

Impaired wound healing states lead to substantial morbidity and cost with treatment resulting in an expenditure of billions of dollars per annum in the U.S. alone. Both chronic wounds and impaired acute wounds are characterized by excessive inflammation, enhanced proteolysis, and reduced matrix deposition. These confounding factors are exacerbated in the elderly, in part, as we report here, related to increased local and systemic tumor necrosis factor-alpha (TNF-α) levels. Moreover, we have used a secretory leukocyte protease inhibitor (SLPI) null mouse model of severely impaired wound healing and excessive inflammation, comparable to age-related delayed human healing, to demonstrate that topical application of anti-TNF-α neutralizing antibodies blunts leukocyte recruitment and NFκB activation, alters the balance between M1 and M2 macrophages, and accelerates wound healing. Following antagonism of TNF-α, matrix synthesis is enhanced, associated with suppression of both inflammatory parameters and NFκB binding activity. Our data suggest that inhibiting TNF-α is a critical event in reversing the severely impaired healing response associated with the absence of SLPI, and may be applicable to prophylaxis and/or treatment of impaired wound healing states in humans.

Estrogen promotes cutaneous wound healing via estrogen receptor beta independent of its antiinflammatory activities.

Post-menopausal women have an increased risk of developing a number of degenerative pathological conditions, linked by the common theme of excessive inflammation. Systemic estrogen replacement (in the form of hormone replacement therapy) is able to accelerate healing of acute cutaneous wounds in elderly females, linked to its potent antiinflammatory activity. However, in contrast to many other age-associated pathologies, the detailed mechanisms through which estrogen modulates skin repair, particularly the cell type-specific role of the two estrogen receptors, ERalpha and ERbeta, has yet to be determined. Here, we use pharmacological activation and genetic deletion to investigate the role of both ERalpha and ERbeta in cutaneous tissue repair. Unexpectedly, we report that exogenous estrogen replacement to ovariectomised mice in the absence of ERbeta actually delayed wound healing. Moreover, healing in epidermal-specific ERbeta null mice (K14-cre/ERbeta(L2/L2)) largely resembled that in global ERbeta null mice. Thus, the beneficial effects of estrogen on skin wound healing are mediated by epidermal ERbeta, in marked contrast to most other tissues in the body where ERalpha is predominant. Surprisingly, agonists to both ERalpha and ERbeta are potently antiinflammatory during skin repair, indicating clear uncoupling of inflammation and overall efficiency of repair. Thus, estrogen-mediated antiinflammatory activity is not the principal factor in accelerated wound healing.

17beta-estradiol inhibits wound healing in male mice via estrogen receptor-alpha.

Although estrogens have long been known to accelerate healing in females, their roles in males remain to be established. To address this, we have investigated the influence of 17beta-estradiol on acute wound repair in castrated male mice. We report that sustained exposure to estrogen markedly delays wound re-epithelialization. Our use of hairless mice revealed this response to be largely independent of hair follicle cycling, whereas other studies demonstrated that estrogen minimally influences wound inflammation in males. Additionally, we report reduced collagen accumulation and increased gelatinase activities in the wounds of estrogen-treated mice. Increased wound matrix metalloproteinase (MMP)-2 activity in these animals may i) contribute to their inability to heal skin wounds optimally and ii) stem, at least in part, from effects on the overall levels and spatial distribution of membrane-type 1-MMP and tissue inhibitor of MMP (TIMP)-3, which respectively facilitate and prevent MMP-2 activation. Using mice rendered null for either the alpha or beta isoform of the estrogen receptor, we identified estrogen receptor-alpha as the likely effector of estrogen's inhibitory effects on healing.

The phytoestrogen genistein promotes wound healing by multiple independent mechanisms.

Genistein has been implicated in the beneficial effects of soy on human health, particularly in the context of ageing. In post-menopausal women reduced systemic estrogen leads to a range of age-associated pathologies, including delayed cutaneous wound healing. We have previously shown that this can be reversed by estrogen replacement. However, the effect of genistein on the skin is poorly understood and crucially the influence of genistein on wound healing has not been assessed. 10-week-old ovariectomised mice were systemically treated with 17beta-estradiol or genistein. Genistein substantially accelerated wound repair, associated with a dampened inflammatory response. Unexpectedly, co-treatment with the ER antagonist ICI had little impact on the anti-inflammatory, healing promoting effects of genistein. Thus genistein's actions are only partially mediated via classical estrogen receptor-dependent signalling pathways. Indeed, we report that alternative (cell-type specific) signalling mechanisms are activated in the skin in response to genistein treatment.

Novel locally active estrogens accelerate cutaneous wound healing. A preliminary study.

New 17beta-estradiol (E2) derivatives 1-11 were synthesized from an estrone derivative by addition of organometallic reagents prepared from protected alpha,omega-alkynols and further elaboration of the addition products. The estrogenic activity of these novel compounds was determined using in vitro binding competition assay and transactivation analysis. Among the E2 derivatives synthesized, compound 2 showed the highest transactivation potency and was therefore tested for its ability to modulate cutaneous wound healing in vivo. Compound 2's ability to accelerate wound healing in ovariectomized mice and decrease the production of inflammatory molecules was comparable to that of E2. However, the activity of compound 2 was not superimposable to E2 with regard to the cells involved in the wound repairing process. When locally administered, compound 2 did not show any systemic activity on ER. This class of compounds with clear beneficial effects on wound healing and suitable for topical administration may lead to the generation of innovative drugs for an area of unmet clinical need.

Unique and synergistic roles for 17beta-estradiol and macrophage migration inhibitory factor during cutaneous wound closure are cell type specific.

The cutaneous wound healing response is complex, comprising numerous overlapping events including inflammation, fibroblast migration, reepithelialization, and wound contraction. With increased age and resultant reduced systemic estrogens, these processes are disrupted and delayed healing ensues. We have demonstrated previously that the proinflammatory cytokine macrophage migration inhibitory factor (MIF) acts as a global regulator of wound healing mediating the majority of estrogen's healing promoting activity. MIF is expressed by numerous wound cell types yet the interaction between estrogens and MIF at the cellular level is still poorly understood. In this study we demonstrate novel accelerated healing in MIF null mice using an excisional wound model. Moreover, we show cell-type-specific differences in the effects of 17beta-estradiol and/or MIF on the cellular function of a range of wound cell types in vitro. Intriguingly, 17beta-estradiol is able to promote the migration of all cell types studied indicating a clear role for cell migration in accelerated wound healing.

Effect of estrogen and progesterone on macrophage activation during wound healing.

Age-related impaired wound healing leads to substantial morbidity and mortality along with a large financial burden to health services. There is accumulating evidence that the tissue damage associated with chronic wounds is initiated and propagated by an inappropriately excessive inflammatory response. Research on age-related impaired wound healing suggests that the decline in sex steroid hormones with age may have a substantial influence on the inflammatory response in vivo. Topical and systemic estrogen treatments have shown an increased rate of healing by reducing inflammation, however the underlying mechanisms are little understood. In vitro studies also suggest progesterone may play a role in modulating inflammation. Macrophages are essential mediators of inflammation and wound healing. Macrophages can be activated in a classical or alternative manner in parallel with the T(H)1/T(H)2 dichotomy, respectively. Using a murine incisional wound healing model this study was carried out to investigate the roles of estrogen and progesterone on macrophage activation during the wound healing response. Our findings suggest with a reduction of steroid hormones following ovariectomy, alternatively activated macrophage markers (Fizz1 and Ym1) were reduced, with this effect being reversed with the administration of estrogen or progesterone; suggesting that with the reduction of steroid hormones macrophages are activated in a classical manner, promoting inflammation, whereas estrogen or progesterone are contributing toward macrophage activation in an alternative manner, driving wound repair, angiogenesis, and remodeling.

Sex dimorphism in wound healing: the roles of sex steroids and macrophage migration inhibitory factor.

That endogenous sex steroid hormones profoundly influence the response to cutaneous injury is well established. How they and other factors combine to direct repair in male and female animals is much less well understood. Using a murine incisional wound-healing model, we investigated the roles of circulating sex steroids, macrophage migration inhibitory factor (MIF) (the mediator of delayed healing in ovariectomized animals), and hormone- and MIF-independent factors in controlling repair. We report that d 3 wounds, of comparable size in intact male and female mice, are significantly larger in ovariectomized female animals than in castrated males, suggesting that native sex hormones mask inherent underlying differences in the ways in which males and females respond to wounding. Wound MIF levels were comparable in intact male and female mice but greater in ovariectomized females than castrated males. Furthermore, wound levels of Jun activation domain-binding protein 1 (JAB1), a key factor by which MIF activates intracellular responses, were increased through ovariectomy and greater in ovariectomized females than castrated males. This difference in wound JAB1 levels may underscore the marked sex difference we observed in the responses of MIF knockout mice to the local application of MIF: healing was impaired in ovariectomized females but not castrated males. Separately, systemic treatment with androgens and estrogens yielded contrasting effects on repair in male and female animals. Collectively, the presented data indicate sex divergence in wound healing to be multifaceted, being strongly influenced by MIF and seemingly limited by the combined actions of gonadal steroids.

Estrogen, not intrinsic aging, is the major regulator of delayed human wound healing in the elderly.

BACKGROUND: Multiple processes have been implicated in age-related delayed healing, including altered gene expression, intrinsic cellular changes, and changes in extracellular milieu (including hormones). To date, little attempt has been made to assess the relative contribution of each of these processes to a human aging phenomenon. The objective of this study is to determine the contribution of estrogen versus aging in age-associated delayed human wound healing. RESULTS: Using an Affymetrix microarray-based approach we show that the differences in gene expression between male elderly and young human wounds are almost exclusively estrogen regulated. Expression of 78 probe sets was significantly decreased and 10 probe sets increased in wounds from elderly subjects (with a fold change greater than 7). A total of 83% of down-regulated probe sets and 80% of up-regulated probe sets were estrogen-regulated. Differentially regulated genes were validated at the level of gene and protein expression, with genes identified as estrogen-regulated in human confirmed as estrogen-dependent in young estrogen depleted mice in vivo. Moreover, direct estrogen regulation is demonstrated for three array-identified genes, Sele, Lypd3 and Arg1, in mouse cells in vitro. CONCLUSION: These findings have clear implications for our understanding of age-associated cellular changes in the context of wound healing, the latter acting as a paradigm for other age-related repair and maintenance processes, and suggest estrogen has a more profound influence on aging than previously thought.

Selective estrogen receptor modulators accelerate cutaneous wound healing in ovariectomized female mice.

A lack of systemic hormones in elderly postmenopausal women leads to delayed cutaneous wound healing. This effect can be reversed by systemic or topical estrogen replacement in both humans and rodent models. Over recent years selective estrogen receptor modulators have been developed in an attempt to achieve the beneficial effects of estrogen clinically, while minimizing the detrimental side effects. The effects of selective estrogen receptor modulators on the skin are poorly understood, and the effects on wound healing have not been assessed. In this study we treated 10-wk-old ovariectomized mice with estradiol, tamoxifen (TAM), raloxifene (RAL), or vehicle and examined the effect on healing of full-thickness incisional wounds. Both TAM and RAL substantially accelerate healing, associated with a dampened inflammatory response and altered inflammatory cytokine profile. In vitro TAM and RAL demonstrate antiinflammatory activity comparable to estrogen. These results have significant implications for the clinical modulation of wound healing.

Androgens influence expression of matrix proteins and proteolytic factors during cutaneous wound healing.

Excessive proteolytic activity is a feature of chronic wounds such as venous ulcers, in which resolution of the inflammatory response fails and restorative matrix accumulation is delayed as a consequence. The inflammatory actions of native androgens during the healing of acute skin wounds have lately been characterized. We have now investigated the hypothesis that such activities may impact upon the balance between anabolic and catabolic processes during wound healing. We report that wound deposition of both type I collagen and fibronectin is increased in castrated rats compared with control animals. This response is accompanied by early increases and later decreases in overall wound levels of the key collagenolytic enzymes, matrix metalloproteinase (MMP)-1 and MMP-13. Moreover, the activities of MMP-2 and MMP-9, two further enzymes that contribute to collagen digestion during venous ulceration, were significantly decreased in the wounds of castrated rats. Additional analyses provide evidence that androgens directly stimulate dermal fibroblast collagen production, which supports the suggestion that increased wound collagen deposition in androgen-deprived rats results from reduced matrix degradation (as opposed to enhanced matrix protein biosynthesis). Androgen-mediated dysregulation of the parallel processes of collagen deposition and turnover may underscore the delayed healing of cutaneous wounds in elderly male patients and further contribute to the increased incidence of non-healing wounds in this population.

The hormonal regulation of cutaneous wound healing.

Conditions of impaired wound healing in the elderly are associated with substantial morbidity and mortality and impose a significant financial burden upon the world's health services. The findings of a series of recent studies have served to highlight the contrasting contributions made by sex steroid hormones to the regulation of cutaneous repair processes. Although estrogens accelerate healing, the actions of the "male" sex hormones 5alpha-dihydrotestosterone and testosterone are primarily deleterious. The shift that occurs in the balance between serum estrogen and androgen levels as a normal feature of human aging may therefore have important consequences for fundamental tissue repair processes.

Androgens modulate the inflammatory response during acute wound healing.

Impaired wound healing states in the elderly lead to substantial morbidity and mortality, and a cost to the health services of over 9 billion dollars per annum. In addition to intrinsic ageing processes that per se cause delayed healing, studies have suggested marked differences in wound repair between the sexes. We have previously reported that, castration of male mice results in a striking acceleration of local cutaneous wound healing and dampens the associated inflammatory response. In this study, we report that systemic 5alpha-reductase inhibition, which blocks the conversion of testosterone to its more active metabolite 5alpha-dihydrotestosterone, mimics the effects of castration in a rat model of cutaneous wound healing. The mechanisms underlying the observed effects involve a direct, cell-specific upregulation of pro-inflammatory cytokine expression by macrophages, but not fibroblasts, in response to androgens. Androgens require the transforming growth factor beta signalling intermediate Smad3 to be present in order to influence repair and local pro-inflammatory cytokine levels. That reducing 5alpha-dihydrotestosterone levels through 5alpha-reductase antagonism markedly accelerates healing suggests a specific target for future therapeutic intervention in impaired wound healing states in elderly males.

Macrophage migration inhibitory factor: a central regulator of wound healing.

Age-associated differences in estrogen levels critically modify the cutaneous wound healing response. Using a microarray-based approach, we profiled changes in gene expression within the wounds of mice that were wild type or null for the pro-inflammatory cytokine macrophage migration inhibitory factor (MIF) in the presence or absence of estrogen. This experimental design identified more than 600 differentially expressed genes and established MIF as a key player in the wound healing process, regulating many novel repair/inflammation-associated gene targets. Moreover, MIF affected virtually all of the effects of reduced estrogen on wound repair. In humans, serum and wound levels of MIF increased with age and were strongly down-regulated by estrogen in vivo. Estrogen-regulated MIF transcription in vitro via a nuclear factor kappaB-dependent mechanism. These findings have wide-ranging implications for the many pathophysiological states in which MIF plays an important regulatory role and suggest a potential therapeutic role for MIF in modulating clinical conditions associated with age-related decline in estrogen levels.

The sex steroid precursor DHEA accelerates cutaneous wound healing via the estrogen receptors.

Age-related impaired wound healing states lead to substantial morbidity and cost, with treatment in the USA resulting in an expenditure of over $9 billion per annum. Dehydroepiandrosterone (DHEA) is a ubiquitous adrenal hormone with immunomodulatory properties whose levels decline significantly with advanced age in humans. Conversion of DHEA locally to downstream steroid hormones leads to estrogenic and/or androgenic effects which may be important in age-related skin homeostasis, and which would avoid systemic adverse effects related to estrogen. We report that systemic DHEA levels are strongly associated with protection against chronic venous ulceration in humans. DHEA accelerated impaired healing in an impaired healing model (mice rendered hypogonadal) associated with increased matrix deposition and dampens the exaggerated inflammatory response. Such effects were mediated by local conversion of DHEA to estrogen, acting through the estrogen receptor, and vitro studies suggest a direct effect on specific pro-inflammatory cytokine production by macrophages via mitogen activated kinase (MAP) and phosphatidylinositol 3 (PI3) kinase pathways. In addition, we show that local injection of DHEA accelerates impaired healing in an ageing mouse colony. We suggest that exogenous application of DHEA accelerates impaired wound repair, results which may be applicable to the prophylaxis and treatment of human impaired wound healing states.

The dinucleotide (CA) repeat polymorphism of estrogen receptor beta but not the dinucleotide (TA) repeat polymorphism of estrogen receptor alpha is associated with venous ulceration.

Venous ulcers are the predominant form of chronic wound in the elderly, accounting for around 70% of all cases. The steroid sex hormone estrogen plays a crucial role in normal human skin maintenance and during cutaneous wound repair following injury. Estrogen can reverse age-related impaired wound healing by dampening the inflammatory response and increasing matrix deposition at the wound site. The molecular actions of estrogen are mediated through two nuclear sex steroid hormone receptors, estrogen receptor alpha (ERalpha) and beta (ERbeta). We have conducted a case-control study to investigate whether dinucleotide repeat polymorphisms in the estrogen receptor genes are associated with venous ulceration in the UK Caucasian population. Genomic fragments containing the ERalpha dinucleotide (TA)(n) repeat polymorphism or the ERbeta dinucleotide (CA)(n) repeat polymorphism were amplified by polymerase chain reaction in subject DNA samples and genotyped according to fragment length by capillary electrophoresis. There was no evidence to suggest that the TA repeat polymorphism of ERalpha was associated with venous ulceration. However, the CA*18 allele of the ERbeta CA repeat polymorphism was significantly associated with venous ulceration (n = 120, OR = 1.8, 95% CI = 1.1-2.8, P = 0.02). When the CA repeats alleles were grouped together into either low (L < or = 18) or high (H > 18) numbers of CA repeats, the low (L) repeat allele was significantly associated with venous ulceration (OR = 1.5, 95% CI = 1.0-2.2, P = 0.03). Our results show that a specific ERbeta variant is associated with impaired healing in the elderly, predisposing individuals to venous ulceration.

Late cornified envelope family in differentiating epithelia--response to calcium and ultraviolet irradiation.

The late cornified envelope (LCE) gene cluster within the epidermal differentiation complex on human chromosome one (mouse chromosome three) contains multiple conserved genes encoding stratum-corneum proteins. Within the LCE cluster, genes form "groups" based on chromosomal position and protein homology. We link a recently accepted nomenclature for the LCE cluster (formerly XP5, small proline-rich-like, late-envelope protein genes) to gene structure, groupings, and chromosomal organization, and carry out a pan-cluster quantitative expression analysis in a variety of tissues and environmental conditions. This analysis shows that (i) the cluster organizes into two "skin" expressing groups and a third group with low-level, tissue-specific expression patterns in all barrier-forming epithelia tested, including internal epithelia; (ii) LCE genes respond "group-wise" to environmental stimuli such as calcium levels and ultraviolet (UV) light, highlighting the functional significance of groups; (iii) in response to UV stimulation there is massive upregulation of a single, normally quiescent, non-skin LCE gene; and (iv) heterogeneity occurs between individuals with one individual lacking expression of an LCE skin gene without overt skin disease, suggesting LCE genes affect subtle attributes of skin function. This quantitative and pan-cluster expression analysis suggests that LCE groups have distinct functions and that within groups regulatory diversification permits specific responsiveness to environmental challenge.

Regulatory roles of androgens in cutaneous wound healing.

Although the effects of androgens on wound healing are poorly characterised, the androgen receptor is expressed by inflammatory cells, keratinocytes and fibroblasts during wound healing, suggesting that androgens may regulate inflammatory and/or repair processes. In fact, it appears that endogenous testosterone inhibits wound healing and promotes inflammation since castration of male mice or systemic treatment with the androgen receptor antagonist flutamide accelerates cutaneous wound healing and reduces the inflammatory response. The aim of this review is to summarise our current knowledge about the regulation of tissue repair processes by androgens.

Role of Smad3 in the hormonal modulation of in vivo wound healing responses.

Smad3 is involved in mediating intracellular signaling by members of the transforming growth factor-beta superfamily and plays a critical role in the cellular proliferation, differentiation, migration, and elaboration of matrix pivotal to cutaneous wound healing. Cross-talk between Smad3 and hormone signaling in vitro has been suggested as an important control mechanism regulating cell activities; however, its relevance in vivo is unknown. Here we report that Smad3 plays a role in androgen-mediated inhibition of wound healing but not in the responses to estrogen modulation in vivo. Both wild-type and Smad3 null female mice exhibited delayed healing following ovariectomy, which could be reversed by estrogen replacement. By contrast, castration accelerated healing in wild-type male mice and was reversible by exogenous androgen treatment. Intriguingly, modulation of androgen levels resulted in no discernible perturbation in the healing response in the Smad3 null mice. Mutant monocytes could be lipopolysaccharide stimulated to produce specific pro-inflammatory agents (macrophage monocyte inhibitory factor) in a fashion similar to wild-type cells, but exhibited a muted response to androgen-mediated stimulation while maintaining a normal response to estrogen-induced macrophage inhibitory factor inhibition. These data suggest that Smad3 plays a role in mediating androgen signaling during the normal wound healing response and implicate Smad3 in the modulation of inflammatory cell activity by androgens.