Damian H. Adams

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

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.

Flii neutralizing antibodies improve wound healing in porcine preclinical studies.

Wound healing is an important area of widely unmet medical need, with millions of procedures carried out worldwide which could potentially benefit from a product to improve the wound repair process. Our studies investigating the actin‐remodeling protein Flightless I (Flii) show it to be an important regulator of wound healing. Flii‐deficient mice have enhanced wound healing in comparison to Flii overexpressing mice which have impaired wound healing. For the first time, we show that a Flightless I neutralizing monoclonal antibody (FnAb) therapy is effective in a large animal model of wound repair. Porcine 5 cm incisional and 6.25 cm2 excisional wounds were treated with FnAb at the time of wounding and for two subsequent days. The wounds were dressed in Tegaderm dressings and left to heal by secondary intention for 7 and 35 days, respectively. At the relevant end points, the wounds were excised and processed for histological analysis. Parameters of wound area, collagen deposition, and scar appearance were analyzed. The results show that treatment with FnAb accelerates reepithelialization and improves the macroscopic appearance of early scars. FnAbs have the potential to enhance wound repair and reduce scar formation.

Tropomyosin Regulates Cell Migration during Skin Wound Healing

Precise orchestration of actin polymer into filaments with distinct characteristics of stability, bundling, and branching underpins cell migration. A key regulator of actin filament specialization is the tropomyosin family of actin-associating proteins. This multi-isoform family of proteins assemble into polymers that lie in the major groove of polymerized actin filaments, which in turn determine the association of molecules that control actin filament organization. This suggests that tropomyosins may be important regulators of actin function during physiological processes dependent on cell migration, such as wound healing. We have therefore analyzed the requirement for tropomyosin isoform expression in a mouse model of cutaneous wound healing. We find that mice in which the 9D exon from the TPM3/γTm tropomyosin gene is deleted (γ9D -/-) exhibit a more rapid wound-healing response 7 days after wounding compared with wild-type mice. Accelerated wound healing was not associated with increased cell proliferation, matrix remodeling, or epidermal abnormalities, but with increased cell migration. Rac GTPase activity and paxillin phosphorylation are elevated in cells from γ9D -/- mice, suggesting the activation of paxillin/Rac signaling. Collectively, our data reveal that tropomyosin isoform expression has an important role in temporal regulation of cell migration during wound healing.

Reply: a novel murine model of hypertrophic scarring using subcutaneous infusion of bleomycin.

Background: The development of new therapies for hypertrophic scarring has been hampered by the lack of an appropriate animal model. The authors’ objective was to establish a reproducible murine model of hypertrophic scarring by infusing bleomycin over a prolonged period to stimulate dermal fibroproliferation. Methods: Osmotic pumps filled with 90 &mgr;l of 2.8 mg/ml bleomycin or a control solution (phosphate-buffered saline) were inserted subcutaneously under the dorsal skin of BALB/c mice. The pumps delivered their content at a constant rate of 0.11 &mgr;l/hour for 28 days before mice were euthanized or kept alive for a further 28 days and euthanized at day 56. The resulting lesions were analyzed using histological and immunohistochemical techniques. Results: The lesions displayed histopathological features of hypertrophic scar similar to those observed in humans and had increased cellularity, abnormal collagen I–collagen III ratios, elevated levels of the proscarring cytokine transforming growth factor &bgr;1, and increased numbers of myofibroblasts. The 28-day model displayed features analogous to those of a developing human hypertrophic scar, while the 56-day model was analogous to a mature hypertrophic scar. Conclusions: The bleomycin infusion model stimulates dermal fibroproliferation, creating reproducible murine scars that are comparable to human hypertrophic scars in terms of histological features, collagen content and organization, cellularity, the presence of myofibroblasts, and expression of transforming growth factor &bgr;1. The bleomycin model represents a promising technique for studying scar formation and testing new antiscarring therapies.

Mouse strains for the ubiquitous or conditional overexpression of the Flii gene

The gelsolin related actin binding protein, Flii, is able to regulate wound healing; mice with decreased Flii expression show improved wound healing whereas mice with elevated Flii expression exhibit impaired wound healing. In both mice and humans Flii expression increases with age and amelioration of FLII activity represents a possible therapeutic strategy for improved wound healing in humans. Despite analysis of Flii function in a variety of organisms we know little of the molecular mechanisms underlying Flii action. Two new murine alleles of Flii have been produced to drive constitutive or tissue‐specific expression of Flii. Each strain is able to rescue the embryonic lethality associated with a Flii null allele and to impair wound healing. These strains provide valuable resources for ongoing investigation of Flii function in a variety of biological processes. genesis 49:681–688, 2011.

Flightless I is a key regulator of the fibroproliferative process in hypertrophic scarring and a target for a novel antiscarring therapy

Hypertrophic scarring carries a large burden of disease, including disfigurement, pain and disability. There is currently no effective medical treatment to reduce or prevent hypertrophic scarring. Flightless I (Flii), a member of the gelsolin family of actin remodelling proteins, is an important negative regulator of wound repair.

Native Australian plant extracts differentially induce Collagen I and Collagen III in vitro and could be important targets for the development of new wound healing therapies

Australian native plants have a long history of therapeutic use in indigenous cultures, however, they have been poorly studied scientifically. We analysed the effects of 14 plant derived compounds from the species Pilidiostigma glabrum, Myoporum montanum, Geijera parviflora, and Rhodomyrtus psidioides for their potential wound healing properties by assessing their ability to induce or suppress Collagen I and Collagen III expression in human skin fibroblasts in culture. The compound 7-geranyloxycoumarin was able to significantly increase Collagen I (23.7%, p<0.0002) expression in comparison to control. Significant suppression of Collagen III was observed for the compounds flindersine (11.1%, p<0.02), and (N-acetoxymethyl) flindersine (27%, p<0.00005). The implications of these finding is that these compounds could potentially alter the expression of different collagens in the skin allowing for the potential development of new wound healing therapies and new approaches for treating various skin diseases as well as photo (sun) damaged, and aged skin.

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.