152 research outputs found
Multifunctional roles of the actin-binding protein Flightless I in inflammation, cancer and wound healing
No full textFlightless 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
No full textWound 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
No full textPublished 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
No full textOver 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
Attenuation of Flightless I, an actin-remodelling protein, improves burn injury repair via modulation of transforming growth factor (TGF)-beta 1 and TGF-beta 3
No full textThe definitive version may be found at www.wiley.comBackground: 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.
Objectives: To investigate the function of Flii gene expression in burn injury repair.
Methods: Partial-thickness scald wounds were created on Flii heterozygous (Flii(+/-)), wild-type (WT) and Flii transgenic (Flii(Tg/+)) mice. Burns were assessed using histology and immunohistochemistry, real-time quantitative polymerase chain reaction and biochemical analysis.
Results: Flii expression, while upregulated in burn injuries, was significantly lower in the wounds of Flii(+/-) vs. WT vs. Flii(Tg/+) mice and healing was improved in Flii(+/-) mice with their burns healing faster than WT and Flii(Tg/+). Pro-scarring transforming growth factor (TGF)-beta 1 protein and gene expression were reduced in Flii(+/-) burns while antiscarring TGF-beta 3 was significantly elevated. Anti-alpha-smooth muscle actin (alpha-SMA) was decreased in Flii(+/-) burns suggesting a decrease in contractile myofibroblasts in the developing scars. Although Flii is primarily a nuclear and cytoplasmic protein it is also released by wounded cells. Intradermal injection of Flii-neutralizing antibodies (FliAbs) to WT burn wounds significantly improved their healing, indicating a potential novel approach for treating burns. Decreased TGF-beta 1 and elevated TGF-beta 3 expression were observed in FliAb-treated burns, which may contribute to their observed improvement in healing.
Conclusions: Strategies aimed at reducing Flii expression, for example using neutralizing antibodies, may lead to improved burn outcomes.D.H. Adams, N. Ruzehaji, X.L. Strudwick, J.E. Greenwood, H.D. Campbell, R. Arkell and A.J. Cowi
Overexpression of Flii during murine embryonic development increases symmetrical division of epidermal progenitor cells
No full textData source: Supplementary material, https://doi.org/10.3390/ijms22158235Epidermal 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
Systemic delivery of anti-integrin alpha L antibodies reduces early macrophage recruitment, inflammation, and scar formation in murine burn wounds
No full textOnline 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
Flightless I expression enhances murine claw regeneration following digit amputation
No full textLink to a related website: http://www.jidonline.org/article/S0022202X16323533/pdf, Open Access via UnpaywallThe mammalian digit tip is capable of both reparative and regenerative wound healing dependent on the level of amputation injury. Removal of the distal third of the terminal phalange results in successful regeneration, whereas a more severe, proximal, amputation heals by tissue repair. Flightless I (Flii) is involved in both tissue repair and regeneration. It negatively regulates wound repair but elicits a positive effect in hair follicle regeneration, with Flii overexpression resulting in significantly longer hair fibers. Using a model of digit amputation in Flii overexpressing (FIT) mice, we investigated Flii in digit regeneration. Both wild-type and FIT digits regenerated after distal amputation with newly regenerated FIT claws being significantly longer than intact controls. No regeneration was observed in wild-type mice after severe proximal amputation; however, FIT mice showed significant regeneration of the missing digit. Using a three-dimensional model of nail formation, connective tissue fibroblasts isolated from the mesenchymal tissue surrounding the wild-type and FIT digit tips and cocultured with skin keratinocytes demonstrated aggregate structures resembling rudimentary nail buds only when Flii was overexpressed. Moreover, β-catenin and cyclin D1 expression was maintained in the FIT regenerating germinal matrix suggesting a potential interaction of Flii with Wnt signaling during regeneration
Human multipotent adult progenitor cell-conditioned medium improves wound healing through modulating inflammation and angiogenesis in mice
No full textData source: Supplementary information, https://doi.org/10.1186/s13287-020-01819-zBackground: 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
Quantum Dots and Andreev Reflections in Graphene
No full textGraphene is an exceptionally thin semiconductor that consists of only one atomic layer of carbon atoms. The electrons in graphene live in a strictly two-dimensional (2D) world. In addition to this remarkable 2Dness, it is also peculiar that the behavior of the electrons in graphene is governed by the Dirac equation rather than the well known Schrödinger’s equation, leading to the discovery of several new physics phenomena. Such unusual properties of graphene have stirred up great excitements since it was first isolated in the lab about five years ago. In this thesis, we investigate the low temperature transport properties of the electrons and holes in several graphene based nano-devices. Overall, two topics are explored in this thesis. First we engineer an energy gap in graphene, which is naturally a zero-gap semiconductor, and further form quantum dot devices on the gapped graphene. The low temperature electronic transport properties of the confined electrons are then studied experimentally in such graphene dots. In a second project,we fabricated Josephson junction devices on graphene using a high critical field superconductor as leads. Here the goal is to research on the interactions between the electrons from graphene and the Cooper pairs from the superconductor in the quantum Hall regime.Kavli Institute of Nanoscience DelftApplied Science
- …
