David M. Ansell

Exploring the “Hair Growth–Wound Healing Connection”: Anagen Phase Promotes Wound Re-Epithelialization

When the skin is damaged, a variety of cell types must migrate, proliferate, and differentiate to reform a functional barrier to the external environment. Recent studies have shown that progenitor cells residing in hair follicles (HFs) are able to contribute to this re-epithelialization of wounds in vivo. However, the influence of the hair cycle on wound healing has not previously been addressed. Here, we have exploited spontaneous postnatal hair-cycle synchronicity in mice to systematically examine the influence of the different hair-cycle stages on murine skin wound healing. We report significant acceleration of healing during the anagen phase of HF cycling in vivo, associated with alterations in epithelial, endothelial, and inflammatory cell types. Intriguingly, gene profiling data reveal a clear correlation between the transcription of genes beneficial for wound healing and those upregulated during the anagen phase of the hair cycle in unwounded skin. These findings, which demonstrate a previously unappreciated association between HF cycling and wound healing, reveal numerous molecular correlates for further investigation.

Animal models of wound repair: Are they cutting it?

Current understanding of the complex process of wound repair is based on decades of study. Integral to this understanding has been the use of in vitro and in vivo models to uncover the key molecular players. Now that major wound processes are more fully understood, therapeutic strategies can be developed to manipulate wound repair. Particularly important areas for future research include developing therapies to aid treatment of healing pathologies such as chronic wounds, and manipulating the normal healing processes to drive a more regenerative phenotype in adults. Here, we discuss the benefits and limitations of current animal‐based models and highlight the urgent need for improved predictive preclinical models for wound healing research. We conclude by suggesting directions where more robust models of chronic wound pathologies may arise, expediting the development of novel therapies.

Direct evidence that PKCα positively regulates wound re-epithelialization: correlation with changes in desmosomal adhesiveness.

Non‐healing wounds cause considerable patient morbidity and represent a significant economic burden. Central to wound repair is re‐epithelialization, a crucial process involving the modulation of cell adhesion to allow keratinocyte migration to cover the exposed underlying tissues. The cellular mechanisms regulating the earliest stages of re‐epithelialization are unclear. We present the first direct evidence that protein kinase Cα (PKCα) plays an important role in regulating wound re‐epithelialization. In PKCα−/− mice re‐epithelialization is delayed, while in novel bitransgenic mice over‐expressing constitutively active PKCα it is accelerated. These effects are not due to changes in keratinocyte proliferation, apoptosis or intrinsic cell motility. Instead, they correlate with changes in desmosomal adhesiveness, delay being preceded by retained desmosomal hyper‐adhesiveness and acceleration with a rapid switch to desmosomal Ca2+‐dependence. We demonstrate mechanistic conservation in acute human wounds where PKCα localizes to wound edge desmosomes, which become Ca2+‐dependent. However, in chronic wounds PKCα remains cytoplasmic and desmosomes fail to switch from the hyper‐adhesive state. These results throw new mechanistic light on the earliest stages of wound re‐epithelialization and suggest activation of PKCα as a new therapeutic strategy for non‐healing wounds. Copyright

A statistical analysis of murine incisional and excisional acute wound models

Mice represent the most commonly used species for preclinical in vivo research. While incisional and excisional acute murine wound models are both frequently employed, there is little agreement on which model is optimum. Moreover, current lack of standardization of wounding procedure, analysis time point(s), method of assessment, and the use of individual wounds vs. individual animals as replicates makes it difficult to compare across studies. Here we have profiled secondary intention healing of incisional and excisional wounds within the same animal, assessing multiple parameters to determine the optimal methodology for future studies. We report that histology provides the least variable assessment of healing. Furthermore, histology alone (not planimetry) is able to detect accelerated healing in a castrated mouse model. Perhaps most importantly, we find virtually no correlation between wounds within the same animal, suggesting that use of wound (not animal) biological replicates is perfectly acceptable. Overall, these findings should guide and refine future studies, increasing the likelihood of detecting novel phenotypes while reducing the numbers of animals required for experimentation.

Hair Follicle Bulge Stem Cells Appear Dispensable for the Acute Phase of Wound Re-epithelialization

The cutaneous healing response has evolved to occur rapidly, in order to minimize infection and to re‐establish epithelial homeostasis. Rapid healing is achieved through complex coordination of multiple cell types, which importantly includes specific cell populations within the hair follicle (HF). Under physiological conditions, the epithelial compartments of HF and interfollicular epidermis remain discrete, with K15+ve bulge stem cells contributing progeny for HF reconstruction during the hair cycle and as a basis for hair shaft production during anagen. Only upon wounding do HF cells migrate from the follicle to contribute to the neo‐epidermis. However, the identity of the first‐responding cells, and in particular whether this process involves a direct contribution of K15+ve bulge cells to the early stage of epidermal wound repair remains unclear. Here we demonstrate that epidermal injury in murine skin does not induce bulge activation during early epidermal wound repair. Specifically, bulge cells of uninjured HFs neither proliferate nor appear to migrate out of the bulge niche upon epidermal wounding. In support of these observations, Diphtheria toxin‐mediated partial ablation of K15+ve bulge cells fails to delay wound healing. Our data suggest that bulge cells only respond to epidermal wounding during later stages of repair. We discuss that this response may have evolved as a protective safeguarding mechanism against bulge stem cell exhaust and tumorigenesis. Stem Cells 2016;34:1377–1385

Re-Evaluating Cyclosporine A as a Hair Growth–Promoting Agent in Human Scalp Hair Follicles

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Oestrogen promotes healing in a bacterial LPS model of delayed cutaneous wound repair.

Wound infection is a major clinical problem, yet understanding of bacterial host interactions in the skin remains limited. Microbe-derived molecules, known as pathogen-associated molecular patterns, are recognised in barrier tissues by pattern-recognition receptors. In particular, the pathogen-associated molecular pattern, lipopolysaccharide (LPS), a component of microbial cell walls and a specific ligand for Toll-like receptor 4, has been widely used to mimic systemic and local infection across a range of tissues. Here we administered LPS derived from Klebsiella pneumoniae, a species of bacteria that is emerging as a wound-associated pathogen, to full-thickness cutaneous wounds in C57/BL6 mice. Early in healing, LPS-treated wounds displayed increased local apoptosis and reduced proliferation. Subsequent healing progression was delayed with reduced re-epithelialisation, increased proliferation, a heightened inflammatory response and perturbed wound matrix deposition. Our group and others have previously demonstrated the beneficial effects of 17β-estradiol treatment across a range of preclinical wound models. Here we asked whether oestrogen would effectively promote healing in our LPS bacterial infection model. Intriguingly, co-treatment with 17β-estradiol was able to promote re-epithelialisation, dampen inflammation and induce collagen deposition in our LPS-delayed healing model. Collectively, these studies validate K. pneumoniae-derived LPS treatment as a simple yet effective model of bacterial wound infection, while providing the first indication that oestrogen could promote cutaneous healing in the presence of infection, further strengthening the case for its therapeutic use.

The battle of the bulge: Re‐evaluating hair follicle stem cells in wound repair

The hair follicle has an established role in wound re‐epithelialisation, a phenomenon that has been appreciated since at least the first half of the last century. The bulge niche, one location of hair follicle epithelial stem cells has been of particular interest to researchers over recent years, with numerous studies showing its ability to directly contribute to epidermal repair. However, recent work has highlighted other progenitor regions of the hair follicle that appear to act as stem cells during epidermal repair. In addition, several studies within the last 12 months have questioned the importance of the bulge during re‐epithelialisation, producing conflicting literature. Here we provide a new model to demonstrate how several important differences in experimental design between studies could account for these seemingly opposing findings, which may have implications for how future studies are conducted.

Pericytes in wound healing: friend or foe?

David M Ansell and Ander Izeta The Centre for Dermatology Research, Institute of Inflammation and Repair, The University of Manchester, Manchester, UK; Instituto Biodonostia, Hospital Universitario Donostia, San Sebastian, Spain Correspondence: David M Ansell, PhD The Centre for Dermatology Research, Institute of Inflammation and Repair, The University of Manchester, UK, Tel.: +44-161-306-0515, Fax: +44 (0)161 306 0693, e-mail: david.ansell@manchester.ac.uk

A guide to studying human dermal adipocytes in situ

Dermal white adipose tissue (DWAT) is a main component of human skin, composed of individual lipid‐laden mesenchymal cells known as dermal adipocytes (DAs). Besides their well‐known role in lipid storage and release, DAs also promote skin immunity, wound healing and hair follicle cycling and are important players in cutaneous neuroendocrinology. The ever‐growing insights into DWAT functions, albeit mostly in mice, have invited speculation that it may be involved in multiple skin diseases ranging from fibrosis to alopecia and psoriasis, thus designating human DWAT a clinically relevant, but as yet insufficiently investigated skin compartment. Therefore, this practical, user‐friendly guide aims to introduce the techniques available to study human DWAT in situ and ex vivo, including immunohistochemistry, immunofluorescence microscopy and analysis via quantitative immunohistomorphometry. Here, we provide information on a collection of stains comprising pre‐adipocyte (Pref1) and mature adipocyte markers (Perilipin1, Caveolin1), as well as various lipid (OilRedO, BODIPY) and histochemical stains (H&E, trichrome) available for use on human DWAT. We offer the reader guidelines on fixing, processing and staining human DAs and highlight caveats and solutions to common problems that one may encounter when studying this fascinating skin compartment. We also suggest standard methods for conducting quantitative immunohistomorphometry on human DWAT and its individual adipocytes to quantify cell size, number, lipid content and fluorescence intensity of adipose‐specific markers. Finally, we briefly introduce in situ hybridization, transmission electron microscopy and essentials of magnetic resonance imaging imaging as additional tools for instructively interrogating this largest, but still least‐known compartment of human skin.