Gillian S. Ashcroft

Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response.

The generation of animals lacking SMAD proteins, which transduce signals from transforming growth factor-β (TGF-β), has made it possible to explore the contribution of the SMAD proteins to TGF-β activity in vivo. Here we report that, in contrast to predictions made on the basis of the ability of exogenous TGF-β to improve wound healing, Smad3-null (Smad3ex8/ex8) mice paradoxically show accelerated cutaneous wound healing compared with wild-type mice, characterized by an increased rate of re-epithelialization and significantly reduced local infiltration of monocytes. Smad3ex8/ex8 keratinocytes show altered patterns of growth and migration, and Smad3ex8/ex8 monocytes exhibit a selectively blunted chemotactic response to TGF-β. These data are, to our knowledge, the first to implicate Smad3 in specific pathways of tissue repair and in the modulation of keratinocyte and monocyte function in vivo.

Conversion of proepithelin to epithelins: roles of SLPI and elastase in host defense and wound repair.

Increased leukocyte elastase activity in mice lacking secretory leukocyte protease inhibitor (SLPI) leads to impaired wound healing due to enhanced activity of TGFbeta and perhaps additional mechanisms. Proepithelin (PEPI), an epithelial growth factor, can be converted to epithelins (EPIs) in vivo by unknown mechanisms with unknown consequences. We found that PEPI and EPIs exert opposing activities. EPIs inhibit the growth of epithelial cells but induce them to secrete the neutrophil attractant IL-8, while PEPI blocks neutrophil activation by tumor necrosis factor, preventing release of oxidants and proteases. SLPI and PEPI form complexes, preventing elastase from converting PEPI to EPIs. Supplying PEPI corrects the wound-healing defect in SLPI null mice. Thus, SLPI/elastase act via PEPI/EPIs to operate a switch at the interface between innate immunity and wound healing.

Secretory leukocyte protease inhibitor mediates non-redundant functions necessary for normal wound healing.

Secretory leukocyte protease inhibitor (SLPI) is a serine protease inhibitor with anti-microbial properties found in mucosal fluids. It is expressed during cutaneous wound healing. Impaired healing states are characterized by excessive proteolysis and often bacterial infection, leading to the hypothesis that SLPI may have a role in this process. We have generated mice null for the gene encoding SLPI (Slpi), which show impaired cutaneous wound healing with increased inflammation and elastase activity. The altered inflammatory profile involves enhanced activation of local TGF-β in Slpi-null mice. We propose that SLPI is a pivotal endogenous factor necessary for optimal wound healing.

Topical Estrogen Accelerates Cutaneous Wound Healing in Aged Humans Associated with an Altered Inflammatory Response

The effects of intrinsic aging on the cutaneous wound healing process are profound, and the resulting acute and chronic wound morbidity imposes a substantial burden on health services. We have investigated the effects of topical estrogen on cutaneous wound healing in healthy elderly men and women, and related these effects to the inflammatory response and local elastase levels, an enzyme known to be up-regulated in impaired wound healing states. Eighteen health status-defined females (mean age, 74.4 years) and eighteen males (mean age, 70.7 years) were randomized in a double-blind study to either active estrogen patch or identical placebo patch attached for 24 hours to the upper inner arm, through which two 4-mm punch biopsies were made. The wounds were excised at either day 7 or day 80 post-wounding. Compared to placebo, estrogen treatment increased the extent of wound healing in both males and females with a decrease in wound size at day 7, increased collagen levels at both days 7 and 80, and increased day 7 fibronectin levels. In addition, estrogen enhanced the strength of day 80 wounds. Estrogen treatment was associated with a decrease in wound elastase levels secondary to reduced neutrophil numbers, and decreased fibronectin degradation. In vitro studies using isolated human neutrophils indicate that one mechanism underlying the altered inflammatory response involves both a direct inhibition of neutrophil chemotaxis by estrogen and an altered expression of neutrophil adhesion molecules. These data demonstrate that delays in wound healing in the elderly can be significantly diminished by topical estrogen in both male and female subjects.

Estrogen modulates cutaneous wound healing by downregulating macrophage migration inhibitory factor

Characteristic of both chronic wounds and acute wounds that fail to heal are excessive leukocytosis and reduced matrix deposition. Estrogen is a major regulator of wound repair that can reverse age-related impaired wound healing in human and animal models, characterized by a dampened inflammatory response and increased matrix deposited at the wound site. Macrophage migration inhibitory factor (MIF) is a candidate proinflammatory cytokine involved in the hormonal regulation of inflammation. We demonstrate that MIF is upregulated in a distinct spatial and temporal pattern during wound healing and its expression is markedly elevated in wounds of estrogen-deficient mice as compared with intact animals. Wound-healing studies in mice rendered null for the MIF gene have demonstrated that in the absence of MIF, the excessive inflammation and delayed-healing phenotype associated with reduced estrogen is reversed. Moreover, in vitro assays have shown a striking estrogen-mediated decrease in MIF production by activated murine macrophages, a process involving the estrogen receptor. We suggest that estrogen inhibits the local inflammatory response by downregulating MIF, suggesting a specific target for future therapeutic intervention in impaired wound-healing states.

Androgen receptor-mediated inhibition of cutaneous wound healing

Impaired wound healing states in the elderly lead to substantial morbidity, mortality, and a cost to the US Health Services of over

Ageing and wound healing

9 billion per annum. In addition to intrinsic aging per se causing delayed healing, studies have suggested marked sex-differences in wound repair. We report that castration of male mice results in a striking acceleration of local cutaneous wound healing, and is associated with a reduced inflammatory response and increased hair growth. Using a hairless mouse model, we have demonstrated that testosterone reduction stimulates the healing response not through hair follicle epithelial/mesenchymal cell proliferation, but directly via effects on wound cell populations. We suggest that endogenous testosterone inhibits the cutaneous wound healing response in males and is associated with an enhanced inflammatory response. The mechanisms underlying the observed effects involve a direct upregulation of proinflammatory cytokine expression by macrophages in response to testosterone. Blockade of androgen action systemically, via receptor antagonism, accelerates healing significantly, suggesting a specific target for future therapeutic intervention in impaired wound healing states in elderly males.

Age-related differences in the temporal and spatial regulation of matrix metalloproteinases (MMPs) in normal skin and acute cutaneous wounds of healthy humans

Cutaneous wound healing is a complex process encompassing a number of overlapping events including leukocyte recruitment, matrix deposition, epithelialization, and ultimately resolution of inflammation with the formation of a mature scar. Morbidity associated with age-related delayed wound healing imposes an enormous social and financial burden; unless improved wound care strategies are developed the projected relative and absolute increase in the elderly population will further exacerbate this problem. In recent years insight has been gained into the impact of ageing on cellular and tissue responses, resulting from impaired cytokine signal transduction, unchecked inflammation, an altered balance of protein synthesis and degradation, and subsequent downstream effects on the rate and quality of the wound healing response. Further understanding of the complex interaction between the ageing cell and its microenvironment is essential in order to develop focussed therapeutic strategies to improve healing in the elderly.

Human ageing impairs injury-induced in vivo expression of tissue inhibitor of matrix metalloproteinases (TIMP)-1 and -2 proteins and mRNA

Abstract.Despite the association of increasing age with chronic wound-healing disorders and an impaired rate of healing of acute cutaneous wounds, the role of matrix metalloproteinases (MMPs) is unknown. To determine the spatial and temporal patterns and activities of MMP-1, -2, -3 and -9, 132 healthy humans aged between 19 and 96 years underwent 4-mm punch biopsies followed by wound excision between day 1 and day 180 post-wounding. Wounds showed an age-related increase in MMP-2 and MMP-9 immunostaining from day 3; this was associated with degradation of gelatin as shown by zymograms and with increased proteinase activity as shown by azocoll assays. Distinct spatial localisations for each MMP were observed: MMP-2 was found in epidermal structures; MMP-9 was observed in inflammatory cells up to day 21; MMP-1 was localised to keratinocytes at the wound margin. Normal old skin showed pro-MMP-2 bands on zymography and increased MMP-2 immunostaining. These results indicate that: (1) intrinsic ageing is associated with the up-regulation of MMPs previously associated with chronic wound healing; (2) wound-tissue proteinases are essentially active up to day 21 postwounding; and (3) intrinsic ageing may predispose to tissue breakdown disorders because of MMP-2 up-regulation in normal skin.

Androgens modulate the inflammatory response during acute wound healing

Proteolysis is an essential component of wound healing but, if uncontrolled, it may lead to degradation of the neo‐matrix and a delay in wound repair. Despite numerous reports of impaired wound healing associated with increasing age, the control of proteolysis is completely unknown. Tissue inhibitor of matrix metalloproteinases (TIMP)‐1 and ‐2 inhibit the activity of matrix metalloproteinases and the pattern of regulation of these molecules determines in part the spatial and temporal regulation of proteolytic activity. This study reports on TIMP‐1 and ‐2 protein localization using immunocytochemistry in healing wounds of healthy subjects of different ages from day 1 to 6 months post‐wounding, and has quantified the mRNA levels for both inhibitors using reverse transcriptase‐polymerase chain reaction (RT‐PCR). TIMP‐1 and TIMP‐2 proteins are up‐regulated from 24 h post‐wounding, with a decrease in staining intensity by day 7 for TIMP‐2 and by day 14 for TIMP‐1. Steady‐state mRNA levels for both TIMPs were significantly greater in normal young skin than in aged skin. In the young, there was a significant increase in mRNA expression for TIMP‐1 and ‐2 by day 3 post‐wounding, which decreased by day 14 and had returned to basal levels at day 21. In the wounds of the aged subjects, basal levels were observed for TIMP‐1 and ‐2 at all time‐points. These results suggest that intrinsic cutaneous ageing is associated with reduced levels of TIMP mRNA both in normal skin and during acute wound repair. These levels may be instrumental in dermal tissue breakdown in normal skin, retarded wound healing, and the predisposition of the elderly to chronic wound healing states.