Hormonal Influences on Wound Healing: A Review of Current Experimental Data

Wounds to the skin heal via a complex series of overlapping stages involving numerous cell and tissue types.¹ In young individuals, these events are tightly regulated. However, in elderly subjects, this regulation and synchronization becomes disrupted. Evidence exists that sex hormones have a modulatory function in a range of biological systems. Differences in the timing and quality of cutaneous healing between genders are strongly indicative of hormonal regulation.^2,3 Aging, Hormones, and Healing Aging is accompanied by a reduction in systemic and local hormone levels. In postmenopausal women, this reduction is swift and dramatic. A clear correlation exists between estrogen levels and the rate of healing with retarded cutaneous healing in post-menopausal women reversed by exogenous estrogen.² The skin has recently been shown to be a steroidogenic tissue containing the full cytochrome P450 system required for the de novo production of sex steroids from cholesterol (Figure 1).⁴ This raises the possibility that bioactive hormones locally synthesized within the wound microenvironment may also be important in this biological system. Increased age and the associated reduction in hormone levels are significant risk factors for development of a nonhealing skin wound, such as a venous ulcer.⁵ Impaired wound healing states—acute wounds that fail to heal and chronic ulcers—are characterized by excessive leukocytosis and subsequently enhanced proteolytic degradation of matrix constituents.^6–8 A full discussion of the underlying pathology and treatment modalities for nonhealing wounds is beyond the scope of this article.⁹ Estrogen Accelerates Healing Age-related changes in skin structure are most prominent in postmenopausal women, ie, due to low estrogen levels. While estrogens are used extensively in hormone replacement therapy (HRT), their mechanisms of action in the skin are only now beginning to be understood. A wide range of cutaneous cell types (eg, fibroblast, endothelial, epithelial, and inflammatory) express estrogen receptors, indicating potential estrogen responsiveness (G.S. Ashcroft, MA, MRCP, PhD, unpublished data 2003). In normal unwounded skin, topical estrogen treatment to some degree reverses 3 age-related skin conditions: 1) skin atrophy by stimulating keratinocyte proliferation and reducing apoptosis, increasing dermal collagen production, and inhibiting MMP expression; 2) skin dryness by altering keratinocyte function, increasing dermal water holding capacity, and increasing sebum production; and 3) skin wrinkles by increasing dermal water holding capacity and increasing number and improving orientation of elastin fibers.¹⁰ Ashcroft et al.^11,12 have previously demonstrated a clear inverse correlation between age and efficiency of acute wound healing. Elderly subjects heal more slowly, and wounds are characterized by increased inflammation, delayed re-epithelization, delayed neovascularization, and reduced matrix deposition due to attenuated fibroblast function.^11,12 The authors’ recent in-vivo and in-vitro studies have begun to characterize the role of hormones in cutaneous healing. In women, a profound shift in healing ability correlates with a dramatic reduction in sex steroids post-menopause. Numerous local proinflammatory cytokines and growth factors are upregulated, and the rate of wound healing declines. The early inflammatory response (neutrophil response) is increased in aged wounds, and macrophage infiltration is delayed.^13,14 Quality of scarring in these estrogen-deprived wounds is substantially improved and associated with reduced local TGF-β1 levels. Systemic HRT significantly accelerates acute wound healing in the elderly, and topical estrogen treatment accelerates the rate of healing in both men and women (Figure 2).^2,15 Ovariectomized (ovx) mice, by virtue of negligible systemic estrogen, provide an excellent experimental model of human age-associated delayed healing (Table 1).¹⁵ Wounds in these mice take substantially longer to heal and contain significantly increased numbers of inflammatory cells compared to intact mice.¹¹ Topical and systemic estrogen treatment accelerates wound healing in ovx mice. Estrogen acts on multiple cell types in the wound to modulate all stages of the healing process (Table 1 and Figure 2). Estrogen has profound effects on the inflammatory phase of healing. Clear differences in the temporal profile of wound inflammatory cell response are apparent between men and women. Estrogen impairs neutrophil chemotaxis, reducing the rate of migration to the wound site while increasing phagocytic function. The net result is more efficient clearance of debris with a net reduction in inflammatory cell-derived protease activity, which indirectly leads to enhanced matrix deposition. Estrogen affects macrophages by reducing production of a range of pro-inflammatory cytokines, including interleukin 6 (IL6),¹⁶ tumor necrosis factor-α (TNF-α), and macrophage migration inhibitory factor (MIF).¹⁷ Estrogen is mitogenic for keratinocytes, accelerating re-epithelization and fibroblasts, increasing synthesis of numerous extracellular matrix components, particularly collagen. While in-vitro experiments suggest the potential of estrogen to accelerate angiogenesis via increased expression of vascular endothelial growth factor (VEGF) and other factors, in-vivo data has proved contradictory. In most inflammatory cells, estrogen-mediated effects on cytokine expression are thought to be regulated at the promoter level.¹⁸ These classical genomic effects involve estrogen binding to estrogen receptors (ERα and/or β), which dimerize and complex with transcription factors, most notably NFkB, an important regulator of pro-inflammatory responses, which can be inhibited by estrogen in vivo. Estrogen is thought to act through differential mechanisms to block NFkB signalling in the cytoplasm and in the nucleus.¹⁸ Alternatively, estrogen and other sex hormones can mediate nongenomic effects, such as rapid induction of protein kinases or other secondary effectors to regulate gene expression.¹⁹ It is likely that the way a given tissue responds to hormone exposure is dependent upon nuclear hormone receptor/splice variant expression profile. In the skin, for example, fibroblasts and keratinocytes express androgen receptor (AR), progesterone receptor (PR), and estrogen receptor β (ERβ).^17,20,21 However, while fibroblasts express ERα, keratinocytes do not. Importantly, human genetic studies in the authors’ lab have recently demonstrated a strong correlation between specific polymorphisms in the ERβ (and not ERα) genes and predisposition to venous ulcer development.²² Since receptor expression and/or function is likely to be affected, topical estrogen treatment would be ineffective in these subjects. In this regard, it is interesting to note that estrogen also appears to protect against development of nonhealing wounds. In a recent case-cohort study, Margolis et al.⁵ identified reduced risk of developing venous leg or pressure ulcers in elderly women undergoing HRT. There may also be a role for progesterone in regulating skin homeostasis and wound healing. In vitro, progesterone stimulates keratinocyte migration, suppresses fibroblast expression of a range of matrix metalloproteinases (MMPs), induces tissue inhibitors of matrix metalloproteinases (TIMP) expression, and increases sebum production. Macrophage Migration Inhibitory Factor Macrophage migration inhibitory factor, a small proinflammatory cytokine identified as a central regulator of innate immunity and inflammation,²³ has been implicated in a host of disease states including tumorigenesis, arthritis, atherosclerosis, and septic shock; it is also associated with tissue injury.²⁴ Using animals genetically null for the MIF gene, the authors have recently identified an important role for MIF in wound healing.¹⁷ In contrast to the delayed healing phenotype observed in ovx wild-type mice, ovx MIF null mice heal normally, ie, the null mouse wounds are irresponsive to estrogen. Now, employing a microarray-based experimental approach, the authors have confirmed MIF as a key downstream mediator of estrogen’s effects on healing. The data highlight the involvement of MIF in the hormonal modulation of all aspects of the healing process.²⁵ Indeed, the findings suggest that estrogen regulates healing almost exclusively via MIF down-regulation and identifies novel MIF-regulated gene targets and clusters associated with aberrant healing (Figure 3). In humans, MIF is upregulated systemically and locally with increasing age. MIF levels are high in chronic nonhealing ulcers then fall with successful healing (M.J. Hardman, PhD, and G.S. Ashcroft, MA, MRCP, PhD, unpublished data 2004). In vitro, estrogen directly regulates MIF at the level of transcription by an estrogen receptor mediated and NFkB-dependent mechanism.^17,25 MIF regulates a plethora of wound healing-associated genes independently of estrogen, indicating a fundamental regulatory role for this cytokine.²⁵ These findings present MIF as an attractive target for potential therapeutic modulation to reverse human age-associated impaired healing. This is of great importance in view of the findings that ERβ function may be disrupted in ulcer subjects and in whom estrogen therapy may be ineffective. In this case, potential modulators of ERβ functions, such as MIF, will be powerful candidates for clinical manipulation. Androgens Retard Healing The male sex hormones, collectively termed androgens, also regulate multiple aspects of skin function (Table 1 and Figure 2).²⁶ Sebaceous gland development and differentiation, hair growth and cycling, and epidermal barrier ontogeny and homeostasis are all androgen-modulated processes. The authors have recently shown that, in contrast to estrogen, androgens inhibit healing. Castrated mice with reduced systemic testosterone display accelerated healing.²⁰ Testosterone appears to modulate healing by directly altering wound cell populations and cytokine profiles, thereby enhancing the inflammatory response and reducing matrix deposition. This is supported by androgen receptor (AR) expression in keratinocytes, fibroblasts, and inflammatory cells within the wound. In vitro, testosterone enhances MIF and TNF expression in lipopolysaccharide (LPS)-activated murine macrophages²⁰ in an AR-dependent manner. The AR antagonist flutamide accelerates wound healing in male mice and suppresses testosterone-induced enhancement of TNF by macrophages. A role for the transcription factor Smad3 in androgen-modulated healing is likely, as Smad3 null mice are insensitive to androgen-induced delayed healing.²⁷ The role of androgens in human healing is highlighted by in-vivo studies demonstrating a positive correlation between increased testosterone levels and delayed healing²⁰ and neural network studies indicating that elderly men are more likely to develop nonhealing ulcers than elderly women.²⁸ Future studies and clinical trials are necessary to confirm the potential of androgen/AR blockade as a viable therapeutic strategy. Dehydroepiandrosterone (DHEA) Dehydroepiandrosterone (DHEA), the predominant adrenal precursor of androgens and estrogens (Figure 1), also progressively declines with age.²⁹ There are numerous lines of evidence to suggest that high serum DHEA levels have a beneficial effect on longevity and prevent a number of human pathophysiological conditions, such as heart disease and diabetes. Using the ovx mouse model of delayed healing, the authors recently demonstrated substantial acceleration of healing following topical DHEA treatment associated with a dampened local inflammatory response and reduced tissue cytokine levels.³⁰ Dehydroepiandrosterone exerts these effects primarily via aromatase conversion to estrogen and subsequent signalling through the estrogen receptor. In vitro, DHEA directly inhibited expression of the pro-inflammatory cytokines IL-6, MIF, and TNF-α through a mechanism involving mitogen activated protein (MAP) and phosphatidyl inositol 3 (PI3) kinase signalling pathways. Finally, DHEA substantially accelerated healing in mice from an aging colony (30 months old) and increased collagen deposition. Dehydroepiandrosterone represents an exciting candidate for clinical manipulation. Its mode of action in the healing wound appears to mimic that of estrogen, yet it has few, if any, of the detrimental systemic side effects of estrogen therapy. Conclusions Hormones exert clear and substantial effects on wound healing. As clinicians begin to develop an understanding of the role of hormones in the healing wound, they can begin to address the cause of the delayed or nonhealing wounds that are experienced by many in the elderly population. Arguably, estrogen is the most important hormone to regulate skin homeostasis and has clear implications for wound healing. Unfortunately, the possible detrimental effect of estrogen treatment on other systems is problematic, highlighted by the recent women’s health initiative studies³¹ and the recent addition of estrogen to the US Department of Health and Human Services’ list of cancer-causing agents. For this reason, the authors are actively identifying and pursuing downstream genes/factors that mediate the effects of estrogen on healing, such as MIF. Moreover, genetic studies suggest that venous ulceration is strongly associated with polymorphisms in the ERβ gene, leading to reduced receptor function. This suggests that therapeutically topical estrogen would be ineffective, necessitating alternative treatments based on the downstream effectors of ERβ signalling. Clinical manipulation of these factors should accelerate healing of acute wounds and aid in the healing of nonhealing wounds, such as venous ulcers, without the detrimental effects of systemic estrogen treatment on other physiological processes.