The influence of Flightless I on toll-like-receptor-mediated inflammation in a murine model of diabetic wound healing

Impaired wound healing and ulceration represent a serious complication of both type 1 and type 2 diabetes. Cytoskeletal protein Flightless I (Flii) is an important inhibitor of wound repair, and reduced Flii gene expression in fibroblasts increased migration, proliferation, and adhesion. As such it has the ability to influence all phases of wound healing including inflammation, remodelling and angiogenesis. Flii has the potential to modulate inflammation through its interaction with MyD88 which it an adaptor protein for TLR4. To assess the effect of Flii on the inflammatory response of diabetic wounds, we used a murine model of streptozocin-induced diabetes and Flii genetic mice. Increased levels of Flii were detected in Flii transgenic murine wounds resulting in impaired healing which was exacerbated when diabetes was induced. When Flii levels were reduced in diabetic wounds of Flii-deficient mice, healing was improved and decreased levels of TLR4 were observed. In contrast, increasing the level of Flii in diabetic mouse wounds led to increased TLR4 and NF-κB production. Treatment of murine diabetic wounds with neutralising antibodies to Flii led to an improvement in healing with decreased expression of TLR4. Decreasing the level of Flii in diabetic wounds may therefore reduce the inflammatory response and improve healing.Nadira Ruzehaji, Stuart J. Mills, Elizabeth Melville, Ruth Arkell, Robert Fitridge and Allison J. Cowi

The role of cytoskeletal protein flightless I (FLII) in diabetic wound healing.

Skin lesions and ulcerations are common and severe complications of diabetes. A significant proportion of these wounds fail to respond to conventional treatment, hence amputation is a feared outcome of diabetes. Overexpression of Flightless (Flii) inhibits wound healing and ablation of Flii using specific neutralising monoclonal antibodies (FnAb) enhances cellular proliferation and migration. It was therefore hypothesized that decreasing Flii expression in diabetic wounds would create a permissive environment for cellular proliferation, enhanced neovascularization, and improved healing outcomes. The aim of this study was to determine whether genetic Flii gene knockdown or treatment with FnAb were effective in improving diabetic wound repair. A mouse model of diabetes was used in which type 1 diabetes was induced using streptozotocin. Diabetes was subsequently induced in low (Flii⁺ʹ⁻), normal (WT) and high (FliiTg/Tg) [Tg/Tg in superscript] mice. Full-thickness dorsal wounds were created and it was found that these wounds healed more rapidly when Flii gene expression was decreased. Further studies revealed that this improved healing was accompanied by a robust pro-angiogenic response with significantly elevated von Willebrand factor and VEGF positive endothelial cell infiltration. In a separate study, wounds in WT diabetic mice were injected intradermally with FnAb and here too improved healing was observed with significantly increased rate of re-epithelialisation compared with placebo control. We investigated the angiogenic response of FnAb both in vitro and in vivo. FnAb enhanced capillary tube formation in human umbilical vein endothelial cells (HUVEC) and promoted formation of functional neovasculature in vivo. Mice with reduced Flii also showed increased numbers of mature blood vessels using an in vivo Matrigel plug assay with increased recruitment of α-SMA positive cells and improved tight junction aiding cell to cell attachments. In conclusion, reducing Flii levels in wounds either genetically or using neutralising antibodies promotes wound healing in diabetic mice by enhancing epithelialisation and improving angiogenic processes. Manipulating Flightless I may therefore be a potential approach for therapeutic intervention in the treatment of the diabetic foot.Thesis (Ph.D.) -- University of Adelaide, School of Paediatrics and Reproductive Health, 201

Burn Wound Management: a surgical perspective

Any patient who survives a large burn injury will be left with some degree of scarring. As well as affecting the form and function of the skin, scarring can have severe psychological consequences such as post-traumatic stress disorder and depression 1. This is particularly the case for hypertrophic or keloid scars, which are common after serious burns. Despite this, the process underlying their formation is incompletely understood and limited effective options are available for their treatment. This paper reviews current understanding of the pathophysiology of the wound healing process in relation to burns and reviews the current management for burn wounds.Cameron AM, Ruzehaji N & Cowin A

Lysosomal secretion of Flightless I upon injury has the potential to alter inflammation

Intracellular Flightless I (Flii), a gelsolin family member, has been found to have roles modulating actin regulation, transcriptional regulation and inflammation. In vivo Flii can regulate wound healing responses. We have recently shown that a pool of Flii is secreted by fibroblasts and macrophages, cells typically found in wounds, and its secretion can be upregulated upon wounding. We show that secreted Flii can bind to the bacterial cell wall component lipopolysaccharide and has the potential to regulate inflammation. We now show that secreted Flii is present in both acute and chronic wound fluid

Attenuation of flightless I improves wound healing and enhances angiogenesis in a murine model of type 1 diabetes

AIMS/HYPOTHESIS: Skin lesions and ulcerations are severe complications of diabetes that often result in leg amputations. In this study we investigated the function of the cytoskeletal protein flightless I (FLII) in diabetic wound healing. We hypothesised that overexpression of FLII would have a negative effect on diabetic wound closure and modulation of this protein using specific FLII-neutralising antibodies (FnAb) would enhance cellular proliferation, migration and angiogenesis within the diabetic wound. METHODS: Using a streptozotocin-induced model of diabetes we investigated the effect of altered FLII levels through Flii genetic knockdown, overexpression or treatment with FnAb on wound healing. Diabetic wounds were assessed using histology, immunohistochemistry and biochemical analysis. In vitro and in vivo assays of angiogenesis were used to assess the angiogenic response. RESULTS: FLII levels were elevated in the wounds of both diabetic mice and humans. Reduction in the level of FLII improved healing of murine diabetic wounds and promoted a robust pro-angiogenic response with significantly elevated von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF)-positive endothelial cell infiltration. Diabetic mouse wounds treated intradermally with FnAb showed improved healing and a significantly increased rate of re-epithelialisation. FnAb improved the angiogenic response through enhanced formation of capillary tubes and functional neovasculature. Reducing the level of FLII led to increased numbers of mature blood vessels, increased recruitment of smooth muscle actin-α-positive cells and improved tight junction formation. CONCLUSIONS/INTERPRETATION: Reducing the level of FLII in a wound may be a potential therapeutic approach for the treatment of diabetic foot ulcers.Nadira Ruzehaji, Zlatko Kopecki, Elizabeth Melville, Sarah L. Appleby, Claudine S. Bonder, Ruth M. Arkell, Robert Fitridge, Allison J. Cowi

Combined effect of genetic background and gender in a mouse model of bleomycin-induced skin fibrosis

International audienceSystemic sclerosis (SSc) is a connective tissue disorder characterised by the development of skin fibrosis. Our current understanding of the disease pathogenesis is incomplete and the study of SSc is hindered, at least partially, by a lack of animal models that fully replicate the complex state of human disease. Murine model of bleomycin-induced dermal fibrosis encapsulates important events that take place early in the disease course.METHODS:To characterise the optimum in vivo parameters required for the successful induction of dermal fibrosis we subjected three commonly used mouse strains to repeated subcutaneous bleomycin injections. We aimed to identify the effects of genetic background and gender on the severity of skin fibrosis. We used male and female Balb/C, C57BL/6, and DBA/2 strains and assessed their susceptibility to bleomycin-induced fibrosis by measuring dermal thickness, hydroxyproline/collagen content and number of resident myofibroblasts, all of which are important indicators of the severity of skin fibrosis. All data are expressed as mean values ± SEM. The Mann-Whitney U test was used for statistical analysis with GraphPad Prism 6.04 software.RESULTS:Dermal fibrosis was most severe in Balb/C mice compared to C57BL/6 and DBA/2 suggesting that Balb/C mice are more susceptible to bleomycin-induced fibrosis. Analysis of the effect of gender on the severity of fibrosis showed that male Balb/C, C57BL/6, DBA/2 mice had a tendency to develop more pronounced fibrosis phenotype than female mice. Of potential importance, male Balb/C mice developed the most severe fibrosis phenotype compared to male C57BL/6 and male DBA/2 as indicated by significantly increased number of dermal myofibroblasts.CONCLUSION:Our study highlights the importance of genetic background and gender in the induction of murine dermal fibrosis. Robust and reproducible animal models of fibrosis are important research tools used in pharmacological studies which may lead to better understanding of the pathogenesis of fibrotic diseases and assist in identification of new drugs

Cytoskeletal protein Flightless (Flii) is elevated in chronic and acute human wounds and wound fluid: neutralizing its activity in chronic but not acute wound fluid improves cellular proliferation

Chronic non-healing wounds form a medical need which will expand as the population ages and the obesity epidemic grows. Whilst the complex mechanisms underlying wound repair are not fully understood, remodelling of the actin cytoskeleton plays a critical role. Elevated expression of the actin cytoskeletal protein Flightless I (Flii) is known to impair wound outcomes. To determine if Flii is involved in the impaired healing observed in chronic wounds, its expression in non-healing human wounds from patients with venous leg ulcers was determined and compared to its expression in acute wounds and unwounded skin. Increased expression of Flii was observed in both chronic and acute wounds with wound fluid and plasma also containing secreted Flii protein. Inflammation is a key aspect of wound repair and fluorescence-activated cell sorting (FACS) analysis revealed Flii was located in neutrophils within the blood and that it co-localised with CD16+ neutrophils in chronic wounds. The function of secreted Flii was investigated as both chronic wound fluid and Flii have previously been shown to inhibit fibroblast proliferation. To determine if the inhibitory effect of wound fluid was due in part to the presence of Flii, wound fluids were depleted of Flii using Flii-specific neutralizing antibodies (FnAb). Flii depleted chronic wound fluid no longer inhibited fibroblast proliferation, suggesting that Flii may contribute to the inhibitory effect of chronic wound fluid on fibroblast function. Application of FnAbs to chronic wounds may therefore be a novel approach used to improve the local environment of non-healing wounds and potentially improve healing outcomes.Nadira Ruzehaji, Randall Grose, Doreen Krumbiegel, Heddy Zola, Pallave Dasari, Hilary Wallace, Michael Stacey, Robert Fitridge, Allison J. Cowi