Xanthe L. Strudwick

Flightless I deficiency enhances wound repair by increasing cell migration and proliferation.

Wound healing disorders are a therapeutic problem of increasing clinical importance involving substantial morbidity, mortality, and rising health costs. Our studies investigating flightless I (FliI), a highly conserved actin‐remodelling protein, now reveal that FliI is an important regulator of wound repair whose manipulation may lead to enhanced wound outcomes. We demonstrate that FliI‐deficient + /− mice are characterized by improved wound healing with increased epithelial migration and enhanced wound contraction. In contrast, FliI‐overexpressing mice have significantly impaired wound healing with larger less contracted wounds and reduced cellular proliferation. We show that FliI is secreted in response to wounding and that topical application of antibodies raised against the leucine‐rich repeat domain of the FliI protein (FliL) significantly improves wound repair. These studies reveal that FliI affects wound repair via mechanisms involving cell migration and proliferation and that FliI might represent an effective novel therapeutic factor to improve conditions in which wound healing is impaired. Copyright

Collagen loss and impaired wound healing is associated with c-Myb deficiency†

Collagen type I serves as an abundant structural and signalling component of skin. It is also an established target gene of the transcription factor, c‐Myb. When c‐myb−/− embryos were examined it was observed that their skin was markedly thinner than normal. Importantly, immunohistochemical investigation showed complete absence of collagen type I. Although these homozygous knock‐out embryos fail to develop beyond day 15, fibroblasts established from these embryos (mouse embryonic fibroblasts [MEFs]) show defective proliferative responses. Furthermore, in vitro scratch wound assays demonstrated that these c‐myb−/− MEFs also exhibit slower closure than their wild‐type counterparts. Embryonic lethality has meant that examination of the role of c‐Myb in adult mouse skin has not been reported to date. However, in view of the abundance of collagen type I in normal skin, its role in skin integrity and the in vitro data showing proliferative and migration defects in c‐myb−/− MEFs, we investigated the consequences of heterozygous c‐myb loss in adult mice on the complex process of skin repair in response to injury. Our studies clearly demonstrate that heterozygous c‐myb deficiency has a functional effect on wound repair, collagen type I levels and, in response to wounding, transforming growth factor‐β1 (an important collagen stimulating factor) induction expression is aberrantly high. Manipulation of c‐Myb may therefore provide new therapeutic opportunities for improving wound repair while uncontrolled expression may underpin some fibrotic disorders. Copyright

Overexpression of the Flii gene increases dermal-epidermal blistering in an autoimmune ColVII mouse model of epidermolysis bullosa acquisita.

Epidermolysis bullosa (EB) is a severe genetic skin fragility syndrome characterized by blister formation. The molecular basis of EB is still largely unknown and wound healing in patients suffering from EB remains a major challenge to their survival. Our previous studies have identified the actin remodelling protein Flightless I (Flii) as an important mediator of wound repair. Here we identify Flii as a novel target involved in skin blistering. Flii expression was significantly elevated in 30 patients with EB, most prominently in patients with recessive dystrophic EB (RDEB) who have defects in production of type VII collagen (ColVII). Using an autoimmune ColVII murine model of EB acquisita (EBA) and an immunocompetent‐ColVII‐hypomorphic genetic mouse model of RDEB together with murine Flii alleles, we investigated the contribution of Flii to EB. Overexpression of Flii produced severe blistering post‐induction of EBA, while decreased Flii reduced blister severity, elevated integrin expression, and improved ColVII production. Flii+/− blistered skin showed reduced α‐SMA, TGF‐β1, and Smad 2/3 expression, suggesting that decreasing Flii may affect fibrosis. In support of this, Flii‐deficient fibroblasts from EBA mice were less able to contract collagen gels in vitro; however, addition of TGF‐β1 restored collagen contraction, suggesting an interplay between Flii and TGF‐β1. Elevated Flii gene and protein expression was further observed in the blisters of ColVII hypomorphic mice, a murine model of RDEB, suggesting that reducing Flii in blistered skin could be a potential new approach for treating patients with EB. Copyright

Attenuation of Flightless I, an actin‐remodelling protein, improves burn injury repair via modulation of transforming growth factor (TGF)‐β1 and TGF‐β3

Background  The pathophysiological mechanisms involved in burn injury repair are still not fully understood but include processes involving cellular proliferation, migration and adhesion. The actin cytoskeleton is intricately involved in these key wound repair processes. Flightless I (Flii), an actin‐remodelling protein and transcriptional regulator, is an important regulator of wound healing.

Gender specific effects on the actin-remodelling protein Flightless I and TGF-β1 contribute to impaired wound healing in aged skin

Impaired wound healing in the elderly presents a major clinical challenge. Understanding the cellular mechanisms behind age-related impaired healing is vital for developing new wound therapies. Here we show that the actin-remodelling protein, Flightless I (FliI) is a contributing factor to the poor healing observed in elderly skin and that gender plays a major role in this process. Using young and aged, wild-type and FliI overexpressing mice we found that aging significantly elevated FliI expression in the epidermis and wound matrix. Aging exacerbated the negative effect of FliI on wound repair and wounds in aged FliI transgenic mice were larger with delayed reepithelialisation. When the effect of gender was further analysed, despite increased FliI expression in young and aged male and female mice, female FliI transgenic mice had the most severe wound healing phenotype suggesting that male mice were refractory to FliI gene expression. Of potential importance, males, but not females, up-regulated transforming growth factor-beta1 and this was most pronounced in aged male FliI overexpressing wounds. As FliI also functions as a co-activator of the estrogen nuclear receptor, increasing concentrations of beta-estradiol were added to skin fibroblasts and keratinocytes and significantly enhanced FliI expression and translocation of FliI from the cytoplasm to the nucleus was observed. FliI further inhibited estrogen-mediated collagen I secretion suggesting a mechanism via which FliI may directly affect provisional matrix synthesis. In summary, FliI is a contributing factor to impaired healing and strategies aimed at decreasing FliI levels in elderly skin may improve wound repair.

IL-5-overexpressing mice exhibit eosinophilia and altered wound healing through mechanisms involving prolonged inflammation

Leucocytes are essential in healing wounds and are predominantly involved in the inflammatory and granulation stages of wound repair. Eosinophils are granulocytic leucocytes and are specifically regulated by interleukin‐5 (IL‐5), a cytokine produced by T helper 2 (Th2) cells. To characterize more clearly the role of the IL‐5 and eosinophils in the wound healing process, IL‐5‐overexpressing and IL‐5‐deficient mice were used as models of eosinophilia and eosinophil depletion, respectively. Our results reveal a significantly altered inflammatory response between IL‐5‐overexpressing and IL‐5 knockout mice post‐wounding. Healing was significantly delayed in IL‐5‐overexpressing mice with wounds gaping wider and exhibiting impaired re‐epithelialization. A delay in collagen deposition was observed suggesting a direct effect on matrix synthesis. A significant increase in inflammatory cell infiltration, particularly eosinophils and CD4+ cells, one of the main cell types which secrete IL‐5, was observed in IL‐5‐overexpressing mice wounds suggesting that one of the main roles of IL‐5 in wound repair may be to promote the infiltration of eosinophils into healing wounds. Healing is delayed in IL‐5‐overexpressing mice and this corresponds to significantly increased levels of eosinophils and CD4+ cells within the wound site that may contribute to and exacerbate the inflammatory response, resulting in detrimental wound repair.

Combination of low calcium with Y-27632 rock inhibitor increases the proliferative capacity, expansion potential and lifespan of primary human keratinocytes while retaining their capacity to differentiate into stratified epidermis in a 3D skin model.

Human keratinocytes are difficult to isolate and have a limited lifespan. Traditionally, immortalised keratinocyte cell lines are used in vitro due to their ability to bypass senescence and survive indefinitely. However these cells do not fully retain their ability to differentiate in vitro and they are unable to form a normal stratum corneum in organotypic culture. Here we aimed to generate a pool of phenotypically similar keratinocytes from human donors that could be used in monolayer culture, without a fibroblast feeder layer, and in 3D human skin equivalent models. Primary human neonatal epidermal keratinocytes (HEKn) were cultured in low calcium, (0.07mM) media, +/-10μM Y-27632 ROCK inhibitor (HEKn-CaY). mRNA and protein was extracted and expression of differentiation markers Keratin 14 (K14), Keratin 10 (K10) and Involucrin (Inv) assessed by qRT-PCR and Western blotting. The differentiation potential of the HEKn-CaY cultures was assessed by increasing calcium levels and removing the Y-27632 for 72hrs prior to assessment of K14, K10 and Inv. The ability of the HEKn-CaY, to form a stratified epithelium was assessed using a human skin equivalent (HSE) model in the absence of Y-27632. Increased proliferative capacity, expansion potential and lifespan of HEKn was observed with the combination of low calcium and 10μM ROCK inhibitor Y-27632. The removal of Y-27632 and the addition of high calcium to induce differentiation allowed the cells to behave as primary keratinocytes even after extended serial passaging. Prolonged lifespan HEK-CaYs were capable of forming an organised stratified epidermis in 3D HSE cultures, demonstrating their ability to fully stratify and retain their original, primary characteristics. In conclusion, the use of 0.07mM Calcium and 10μM Y-27632 in HEKn monocultures provides the opportunity to culture primary human keratinocytes without a cell feeder layer for extended periods of culture whilst retaining their ability to differentiate and form a stratified epithelium.

Cytoskeletal Regulation of Dermal Regeneration

Wound healing results in the repair of injured tissues however fibrosis and scar formation are, more often than not the unfortunate consequence of this process. The ability of lower order vertebrates and invertebrates to regenerate limbs and tissues has been all but lost in mammals; however, there are some instances where glimpses of mammalian regenerative capacity do exist. Here we describe the unlocked potential that exists in mammals that may help us understand the process of regeneration post-injury and highlight the potential role of the actin cytoskeleton in this process. The precise function and regulation of the cytoskeleton is critical to the success of the healing process and its manipulation may therefore facilitate regenerative healing. The gelsolin family of actin remodelling proteins in particular has been shown to have important functions in wound healing and family member Flightless I (Flii) is involved in both regeneration and repair. Understanding the interactions between different cytoskeletal proteins and their dynamic control of processes including cellular adhesion, contraction and motility may assist the development of therapeutics that will stimulate regeneration rather than repair.

How plasma induced oxidation, oxygenation, and de-oxygenation influences viability of skin cells

The effect of oxidation, oxygenation, and de-oxygenation arising from He gas jet and He plasma jet treatments on the viability of skin cells cultured in vitro has been investigated. He gas jet treatment de-oxygenated cell culture medium in a process referred to as “sparging.” He plasma jet treatments oxidized, as well as oxygenated or de-oxygenated cell culture medium depending on the dissolved oxygen concentration at the time of treatment. He gas and plasma jets were shown to have beneficial or deleterious effects on skin cells depending on the concentration of dissolved oxygen and other oxidative molecules at the time of treatment. Different combinations of treatments with He gas and plasma jets can be used to modulate the concentrations of dissolved oxygen and other oxidative molecules to influence cell viability. This study highlights the importance of a priori knowledge of the concentration of dissolved oxygen at the time of plasma jet treatment, given the potential for significant impact on the biol...

In vivo delivery of functional Flightless I siRNA using layer-by-layer polymer surface modification.

Gene silencing using small interfering RNA has been proposed as a therapy for cancer, viral infections and other diseases. This study aimed to investigate whether layer-by-layer polymer surface modification could deliver small interfering RNA to decrease fibrotic processes associated with medical device implantation. Anti-green fluorescent protein labelled small interfering RNA was applied to tissue culture plates and polyurethane using a layer-by-layer technique with small interfering RNA and poly-L-lysine. In vitro studies showed that the level of down-regulation of green fluorescent protein was directly related to the number of coatings applied. This layer-by-layer coating technique was then used to generate Rhodamine-Flii small interfering RNA-coated implants for in vivo studies of small interfering RNA delivery via subcutaneous implantation in mice. After two days, Rh-positive cells were observed on the implants’ surface indicating cellular uptake of the Rhodamine-Flii small interfering RNA. Decreased Flii gene expression was observed in tissue surrounding the Rhodamine-Flii small interfering RNA coated implants for up to seven days post implantation, returning to baseline by day 21. Genes downstream from Flii, including TGF-β1 and TGF-β3, showed significantly altered expression confirming a functional effect of the Rhodamine-Flii small interfering RNA on gene expression. This research demonstrates proof-of-principle that small interfering RNA can be delivered via layer-by-layer coatings on biomaterials and thereby can alter the fibrotic process.