Increased expression of flightless I in cutaneous squamous cell carcinoma affects wnt/beta-catenin signaling pathway

Cutaneous squamous cell carcinoma (cSCC) accounts for 25% of cutaneous malignancies diagnosed in Caucasian populations. Surgical removal in combination with radiation and chemotherapy are effective treatments for cSCC. Nevertheless, the aggressive metastatic forms of cSCC still have a relatively poor patient outcome. Studies have linked actin cytoskeletal dynamics and the Wnt/β-catenin signaling pathway as important modulators of cSCC pathogenesis. Previous studies have also shown that the actin-remodeling protein Flightless (Flii) is a negative regulator of cSCC. The aim of this study was to investigate if the functional effects of Flii on cSCC involve the Wnt/β-catenin signaling pathway. Flii knockdown was performed using siRNA in a human late stage aggressive metastatic cSCC cell line (MET-1) alongside analysis of Flii genetic murine models of 3-methylcholanthrene induced cSCC. Flii was increased in a MET-1 cSCC cell line and reducing Flii expression led to fewer PCNA positive cells and a concomitant reduction in cellular proliferation and symmetrical division. Knockdown of Flii led to decreased β-catenin and a decrease in the expression of the downstream effector of β-catenin signaling protein SOX9. 3-Methylcholanthrene (MCA)-induced cSCC in Flii overexpressing mice showed increased markers of cancer metastasis including talin and keratin-14 and a significant increase in SOX9 alongside a reduction in Flii associated protein (Flap-1). Taken together, this study demonstrates a role for Flii in regulating proteins involved in cSCC proliferation and tumor progression and suggests a potential role for Flii in aggressive metastatic cSCC.Gink N. Yang, Xanthe L. Strudwick, Claudine S. Bonder, Zlatko Kopecki and Allison J. Cowi

Overexpression of Flii during murine embryonic development increases symmetrical division of epidermal progenitor cells

Epidermal progenitor cells divide symmetrically and asymmetrically to form stratified epidermis and hair follicles during late embryonic development. Flightless I (Flii), an actin remodelling protein, is implicated in Wnt/β-cat and integrin signalling pathways that govern cell division. This study investigated the effect of altering Flii on the divisional orientation of epidermal progenitor cells (EpSCs) in the basal layer during late murine embryonic development and early adolescence. The effect of altering Flii expression on asymmetric vs. symmetric division was assessed in vitro in adult human primary keratinocytes and in vivo at late embryonic development stages (E16, E17 and E19) as well as adolescence (P21 day-old) in mice with altered Flii expression (Flii knockdown: Flii(+/-), wild type: WT, transgenic Flii overexpressing: Flii(Tg/Tg)) using Western blot and immunohistochemistry. Flii(+/-) embryonic skin showed increased asymmetrical cell division of EpSCs with an increase in epidermal stratification and elevated talin, activated-Itgb1 and Par3 expression. Flii(Tg/Tg) led to increased symmetrical cell division of EpSCs with increased cell proliferation rate, an elevated epidermal SOX9, Flap1 and β-cat expression, a thinner epidermis, but increased hair follicle number and depth. Flii promotes symmetric division of epidermal progenitor cells during murine embryonic development.Gink N. Yang, Parinaz Ahangar, Xanthe L. Strudwick , Zlatko Kopecki and Allison J. Cowi

Attenuation of flightless I increases human pericyte proliferation, migration and angiogenic functions and improves healing in murine diabetic wounds

Pericytes are peri-vascular mural cells which have an important role in the homeostatic regulation of inflammatory and angiogenic processes. Flightless I (Flii) is a cytoskeletal protein involved in regulating cellular functions, but its involvement in pericyte activities during wound healing is unknown. Exacerbated inflammation and reduced angiogenesis are hallmarks of impaired diabetic healing responses, and strategies aimed at regulating these processes are vital for improving healing outcomes. To determine the effect of altering Flii expression on pericyte function, in vitro and in vivo studies were performed to assess the effect on healing, inflammation and angiogenesis in diabetic wounds. Here, we demonstrated that human diabetic wounds display upregulated expression of the Flii protein in conjunction with a depletion in the number of platelet derived growth factor receptor β (PDGFRβ) +/ neural glial antigen 2 (NG2) + pericytes present in the dermis. Human pericytes were found to be positive for Flii and attenuating its expression in vitro through siRNA knockdown led to enhanced proliferation, migration and angiogenic functions. Genetic knockdown of Flii in a streptozotocin-induced murine model of diabetes led to increased numbers of pericytes within the wound. This was associated with dampened inflammation, an increased rate of angiogenic repair and improved wound healing. Our findings show that Flii expression directly impacts pericyte functions, including proliferation, motility and angiogenic responses. This suggests that Flii regulation of pericyte function may be in part responsible for the changes in pericyte-related processes observed in diabetic wounds.Hannah M Thomas, Parinaz Ahangar, Benjamin R Hofma, Xanthe L Strudwick, Robert Fitridge, Stuart J Mills and Allison J Cowi

Human multipotent adult progenitor cell-conditioned medium improves wound healing through modulating inflammation and angiogenesis in mice

Background: Stem cell therapies have been widely investigated for their healing effects. However, the translation of these therapies has been hampered by the requirement to deliver live allogeneic or autologous cells directly to the wound in a clinical setting. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived adherent stem cells that secrete a wide range of factors known to accelerate the wound healing process. The aim of this study was to determine the impact of MAPC cells secretome on healing outcomes without the presence of MAPC cells. Methods: The effect of MAPC-conditioned medium (MAPC-CM) on the capacity of keratinocytes, fibroblasts and endothelial cells to migrate and proliferate was determined in vitro using scratch wound closure and WST1 assay, respectively. The effect of MAPC-CM on collagen deposition and angiogenesis was also assessed using in vitro methods. Additionally, two excisional wounds were created on the dorsal surface of mice (n = 8/group) and 100 μL of 20× MAPC-CM were intradermally injected to the wound margins. Wound tissues were collected at 3, 7 and 14 days post-wounding and stained with H&E for microscopic analysis. Immunohistochemistry was performed to investigate inflammation, angiogenesis and collagen deposition in the wounds. Results: Skin fibroblasts, keratinocytes and endothelial cells treated with MAPC-CM all showed improved rates of scratch closure and increased cellular proliferation. Moreover, fibroblasts treated with MAPC-CM deposited more collagens I and III and endothelial cells treated with MAPC-CM showed increased capillary tube formation. Murine excisional wounds intradermally injected with MAPC-CM showed a significant reduction in the wound area and an increase in the rate of reepithelialisation. The results also showed that inflammatory cell infiltration was decreased while an increase in angiogenesis, as well as collagens I and III expressions, was observed. Conclusion: These findings suggest that factors produced by MAPC cells can have an important effect on cutaneous wound healing by affecting skin cell proliferation and migration, balancing inflammation and improving the formation of extracellular matrix and angiogenesis. Development of stem cell-free therapy for the treatment of wounds may be a more clinically translatable approach for improving healing outcomes.Parinaz Ahangar, Stuart J. Mills, Louise E. Smith, Xanthe L. Strudwick, Anthony E. Ting, Bart Vaes, and Allison J. Cowi

Multifunctional roles of the actin-binding protein Flightless I in inflammation, cancer and wound healing

Flightless I is an actin-binding member of the gelsolin family of actin-remodeling proteins that inhibits actin polymerization but does not possess actin severing ability. Flightless I functions as a regulator of many cellular processes including proliferation, differentiation, apoptosis, and migration all of which are important for many physiological processes including wound repair, cancer progression and inflammation. More than simply facilitating cytoskeletal rearrangements, Flightless I has other important roles in the regulation of gene transcription within the nucleus where it interacts with nuclear hormone receptors to modulate cellular activities. In conjunction with key binding partners Leucine rich repeat in the Flightless I interaction proteins (LRRFIP)1/2, Flightless I acts both synergistically and competitively to regulate a wide range of cellular signaling including interacting with two of the most important inflammatory pathways, the NLRP3 inflammasome and the MyD88-TLR4 pathways. In this review we outline the current knowledge about this important cytoskeletal protein and describe its many functions across a range of health conditions and pathologies. We provide perspectives for future development of Flightless I as a potential target for clinical translation and insights into potential therapeutic approaches to manipulate Flightless I functions.Xanthe L. Strudwick and Allison J. Cowi

Wound Healing from an Actin Cytoskeletal Perspective

Wound healing requires a complex cascade of highly controlled and conserved cellular and molecular processes. These involve numerous cell types and extracellular matrix molecules regulated by the actin cytoskeleton. This microscopic network of filaments is present within the cytoplasm of all cells and provides the shape and mechanical support required for cell movement and proliferation. Here, an overview of the processes of wound healing are described from the perspective of the cell in relation to the actin cytoskeleton. Key points of discussion include the role of actin, its binding proteins, signaling pathways, and events that play significant roles in the phases of wound healing. The identification of cytoskeletal targets that can be used to manipulate and improve wound healing is included as an emerging area of focus that may inform future therapeutic approaches to improve healing of complex wounds.Parinaz Ahangar, Xanthe L. Strudwick, and Allison J. Cowi

Effect of flightless I expression on epidermal stem cell niche during wound repair

Published Online Ahead of Print:July 3, 2019Objective: Activation of epidermal stem cells (EpSCs) from their quiescent niche is an integral component of wound reepithelialization and involves Wnt/bcatenin (b-Cat) signaling and remodeling of the actin cytoskeleton. The aim of this study was to investigate the effect of Flightless I (Flii), a cytoskeletal protein and inhibitor of wound healing, on EpSC activation during wound repair. Approach: Genetically modified Flii mice (Flii knockdown: Flii+/-, wild type: WT, Flii overexpressing: FliiTg/Tg) received two incisional wounds along the lateral axis of the dorsal skin. Indicators of EpSC activation (epidermal growth factor receptor 1 [EGFR1], leucine-rich repeats and immunoglobulin-like domains-1 [Lrig1], K14), Wnt/b-Cat signaling (Lgr6, Flap2, b-Cat, and axis inhibition protein 2 [Axin2]), and cell proliferation (proliferating cell nuclear antigen [PCNA]) were assessed using immunohistochemistry. b-Cat stabilization was examined using western blotting with cell cycling and differentiation of isolated CD34+ITGA6high EpSCs examined using real time-quantitative polymerase chain reaction after treatment with wound-conditioned media. Results: Flii+/- led to increased numbers of activated EpSCs expressing PCNA, elevated EGFR1, and decreased Lrig1. EpSCs in Flii+/- hair follicle niches adjacent to the wounds also showed expression of Wnt-activation markers including increased b-Cat and Lgr6, and decreased Axin2. EpSCs (CD34+ITGA6high) isolated from Flii+/- unwounded skin showed elevated expression of cell-cycling genes including DNp63, filaggrin (Fila), involucrin (Invo), cyclin D1 (Ccnd1), and cell-division cycle protein-20 (Cdc20); and elevated DNp63 and Invo after treatment with wound-conditioned media compared with WT and FliiTg/Tg counterparts. Innovation: Flii was identified as an inhibitor of EpSC activation that may explain its negative effects on wound reepithelialization. Conclusion: Flii may inhibit EpSC activation by interrupting Wnt/b-Cat signaling. Strategies that reduce Flii may increase activation of EpSCs and promote reepithelialization of wounds.Gink N. Yang, Xanthe L. Strudwick, Claudine Bonder, Zlatko Kopecki, and Allison J. Cowi

The development of microfluidic-based western blotting: Technical advances and future perspectives

Over the past two decades significant technical advancement in the field of western blotting has been made possible through the utilization of microfluidic technologies. In this review we provide a critical overview of these advancements, highlighting the advantages and disadvantages of each approach. Particular attention is paid to the development of now commercially available systems, including those for single cell analysis. This review also discusses more recent developments, including algorithms for automation and/or improved quantitation, the utilization of different materials/chemistries, use of projection electrophoresis, and the development of triBlots. Finally, the review includes commentary on future advances in the field based on current developments, and the potential of these systems for use as pointof-care devices in healthcare.Christopher T. Desire, R. Dario Arrua, Xanthe L. Strudwick, Zlatko Kopecki, Allison J. Cowin, Emily F. Hilde

Systemic delivery of anti-integrin alpha L antibodies reduces early macrophage recruitment, inflammation, and scar formation in murine burn wounds

Online Ahead of Print:January 28, 2020Objective: Increased macrophage recruitment in the early stages of wound healing leads to an excessive inflammatory response associated with elevated fibrosis and scarring. This recruitment relies upon integrins on the surface of monocytes that regulate their migration and extravasation from the circulation into the wound site, where they differentiate into macrophages. The aim of this study was to determine if inhibiting monocyte extravasation from the circulation into burns would reduce macrophages numbers in burns and lead to reduced inflammation and scar formation. Approach: Scald burns were created on mice and treated with integrin alpha L (αL) function blocking antibody via intravenous delivery day 1 after injury. The effect of inhibiting macrophage recruitment into the burn was assessed using macro- and microscopic wound parameters as well as immunohistochemistry for inflammatory cell markers, cytokines, and collagen deposition. Results: Burn wound-associated macrophages were reduced by 54.7% at day 3 following treatment with integrin αL antibody, with levels returning to normal by day 7. This reduction in macrophages led to a concomitant reduction in inflammatory mediators, including tumor necrosis factor-alpha (TNFα) and Il-10 as well as a reduction in proscarring transforming growth factor beta 1 (TGFβ1). This reduced inflammatory response was also associated with less alpha smooth muscle actin (αSMA) expression and an overall trend toward reduced scar formation with a lower collagen I/III ratio. Innovation: Treatment of burns with integrin αL function blocking antibodies reduces inflammation in burn wounds. Conclusion: These results suggest that reducing macrophage infiltration into burn wounds may lead to a reduced early inflammatory response and less scar formation following burn injury.Xanthe L. Strudwick, Damian H. Adams, Natasha T. Pyne, Michael S. Samuel, Rachael Z. Murray and Allison J. Cowi

Treatment of murine partial thickness scald injuries with multipotent adult progenitor cells decreases inflammation and promotes angiogenesis leading to improved burn injury repair

Stem cells have been shown to have potential as a new therapy for burns and promote wound healing through decreasing inflammation and increasing angiogenesis. Multipotent adult progenitor cells (MAPC® cells) are a subpopulation of bone marrow-derived stem cells with outstanding self-renewal and differentiation capacity. MAPC cells also secrete a wide range of cytokines which can affect cellular activities. This article aimed to examine the effects of MAPC cells treatment on burn injury repair using a mouse model of partial thickness burn injury. The immunomodulatory effect of MAPC cells was investigated in vitro using a simultaneous T-cell proliferation assay. Partial thickness burns were created on the dorsal surface of mice and MAPC cells were administered via intradermal injection to the wound margins 24 h post-burn injury. The burn tissues were analysed macroscopically to determine wound area and histologically assessed to determine wound width and rate of re-epithelialisation. Immunohistochemistry and ELISA were employed to assess cell proliferation, inflammation and angiogenesis and collagen deposition in the burn area. MAPC cells inhibit the proliferation of stimulated T cells in culture. Burns intradermally injected with MAPC cells showed a significant reduction in the macroscopic wound area, histologic wound width and had an increased rate of re-epithelialisation. Immunohistochemistry and ELISA analysis of burn tissues showed dampened inflammation evidenced by a reduction in neutrophilic infiltration and modulation of inflammatory cytokines. Angiogenesis within the burn area was also improved in MAPC cell treated mice. However, no significant effect of MAPC cell treatment was observed on extracellular matrix production. Treatment of burns with MAPC cells improved burn injury repair with reduced time to healing, decreased inflammation and increased angiogenesis. These findings demonstrate the promising effects of MAPC cells on burn injury repair and suggest MAPC cells as a candidate source for clinical cell therapies.Parinaz Ahangar, Stuart J. Mills, Louise E. Smith, Xanthe L. Strudwick, Anthony E. Ting, Bart Vaes, Allison J. Cowi