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Ephrin-A2 Affects Wound Healing and Scarring in a Murine Model of Excisional Injury

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Ephrin-A2 Affects Wound Healing and Scarring in a Murine Model of Excisional Injury b u r n s 4 5 ( 2 0 1 9 ) 6 8 2 – 6 9 0 Available online at www.sciencedirect.com ScienceDirect jo urnal homepage: www.elsevier.com/locate/burns Ephrin-A2 affects wound healing and scarring in a murine model of excisional injury a b a Dulharie Wijeratne , Jennifer Rodger , Andrew Stevenson , a c,d a,d,e,f Hilary Wallace , Cecilia M. Prêle , Fiona M. Wood , a,c,e, Mark W. Fear * a Burn Injury Research Unit, School of Biomedical Sciences, The University of Western Australia, Australia b Experimental and Regenerative Neurosciences, School of Biological Sciences, The University of Western Australia, Australia c The Institute for Respiratory Health, The University of Western Australia, Nedlands, Western Australia, Australia d Centre for Cell Therapy and Regenerative Medicine, School of Biomedical Sciences, The University of Western Australia, Crawley, Western Australia, Australia e The Fiona Wood Foundation, Perth, Western Australia, Australia f Burns Service of Western Australia, WA Department of Health, Perth, Western Australia, Australia a r t i c l e i n f o a b s t r a c t Article history: Ephrin ligand/Eph receptor signaling is important in both tissue development and Accepted 4 October 2018 homeostasis. There is increasing evidence that Ephrin/Eph signaling is important in the skin, involved in hair follicle cycling, epidermal differentiation, cutaneous innervation and skin cancer. However, there is currently limited information on the role of Ephrin/Eph signaling in cutaneous wound healing. Here we report the effects of the Ephrin-A2 and À/À À/À À/À Keywords: A5 ligands on wound healing. Using Ephrin-A2 , Ephrin-A5 and Ephrin-A2A5 transgenic mice, in vitro wound healing assays were conducted using isolated keratinocytes Ephrin A2 À/À À/À Collagen and fibroblasts. Ephrin-A2 , Ephrin-A2A5 and wild type mice with excisional wounds Scarring were used to analyze the impact of these ligands on wound closure, scar outcome, collagen orientation and re-innervation in vivo. Wound repair Keratinocyte The absence of the Ephrin-A2 and A5 ligands did not have any effect on dermal fibroblast proliferation or on fibroblast or keratinocyte migration. The loss of Ephrin-A2 and A5 ligands did not impact on the rate of wound closure or re-innervation after injury. However, changes in the gross morphology of the healed scar and in collagen histology of the scar dermis were observed in transgenic mice. Therefore Ephrin-A2 and A5 ligands may play an important role in final scar appearance associated with collagen deposition and structure. © 2018 Elsevier Ltd and ISBI. All rights reserved. * Corresponding author at: Burn Injury Research Unit, M318, University of Western Australia, 35 Stirling Highway, Crawley, WA 6009 Australia. E-mail address: [email protected] (M.W. Fear). https://doi.org/10.1016/j.burns.2018.10.002 0305-4179/© 2018 Elsevier Ltd and ISBI. All rights reserved. b u r n s 4 5 ( 2 0 1 9 ) 6 8 2 – 6 9 0 683 2.2. In vitro scratch assay 1. Introduction À/À Dermal fibroblasts from Wild-type (n=5), Ephrin-A2 (n=5), À/À À/À Eph receptor/Ephrin ligand interactions are important in Ephrin-A5 (n=5) and Ephrin-A2A5 (n=5) mice were neuronal mapping and topography in central and peripheral isolated, cultured and plated in 6 well tissue culture plates. 5 nerves [1]. All the Eph receptors and Ephrin ligands are The seeding density of dermal fibroblasts was 2Â10 /ml of expressed in normal human skin [2] and Ephrin-A ligand Dulbecco’s modified eagle medium nutrient mixture F-12 1 signaling has been shown to be important in hair follicle (DMEM/F12, GIBCO Carlsbad, California, USA) supplemented cycling and epidermal differentiation [3–5]. Ephrin A2 and with 10% fetal bovine serum (FBS, Invitro technologies, Noble 1 A5 are critical for sensory axonal growth patterning and in Park North, Australia) and 1% penicillin/streptomycin (GIBCO the development of cutaneous innervation in animal models by Life Technologies, Carlsbad, California, USA). Plates were – [4 7]. In addition, keratinocytes treated with human Fc- incubated at 37 C in 5% CO2 for the cells to reach full confluence conjugated Ephrin-A ligands have been shown to suppress over two days when a scratch was made as previously described the expression of important cell adhesion genes such as [17]. Photographs were taken at 0, 6, 24, 30 and 48h using a light integrin b6 and integrin b4, suggesting Ephrin signaling may microscope (Olympus IX51, Japan) at a final objective 10Â, influence cell migration through reduced cell attachment numerical aperture 0.13. The total photographed area and the and increased motility [4]. Eph A3 mediated activation of area of the scratch was measured using the ImageJ software Ephrin A2 or A5 signaling has also been shown to result in program [18]. The percentage closure was calculated by increased cell adhesion to laminin through increased subtracting the percentage of the scratch area remaining at b1 integrin expression [8]. Since b1 integrin plays an each time point from the percentage of the scratch at 0h(100%) important role in wound repair [9] this evidence suggests to obtain the percentage closure (% surface area of the scratch Ephrin signaling may be important in skin repair after injury. thatwas coveredbycells). Cellmigrationwas representedasthe Ephrin signaling is also important in skin cancer, with percentage closure of the scratch over time. significant roles in melanoma and more recently demon- strated role in squamous cell carcinoma (SCC), with a key role 2.3. Primary dermal fibroblast proliferation assay for ephrin signaling in migration and invasion of tumour À/À cells [10–12]. Ephrin signaling has also been implicated in the Dermal fibroblasts from wild-type (n=5), Ephrin-A2 (n=5), À/À À/À response to hypoxia in the skin, potentially implicating Ephrin-A5 (n=5) and Ephrin-A2A5 (n=5) mice, were 3 ephrin signaling in angiogenesis in wound repair as well as in seeded at 1.5Â10 cells per well in 96 well microplates in tumours [13]. However, whilst all this evidence points to an 100ml of DMEM/F12 supplemented with 10% FBS and 1% important role for ephrin signaling in wound repair, to date penicillin/streptomycin. Each sample contained 3–5 replicate there have been no studies demonstrating the effects of wells. Control wells contained 100ml of DMEM/ ephrin signaling on scar formation after cutaneous injury in F12 supplemented with 10% FBS and 1% penicillin/streptomy- vivo. We hypothesized that Ephrin A2 and/or A5 signaling cin. Cells were incubated at 37 C with 5% CO2. Separate plates would be important in wound repair. Specifically, with the were used for 24, 48, 72 and 96h incubation times after seeding 1 known role for these ligands in the development of dermal fibroblasts. At each time-point, 20ml of CellTiter 96 cutaneous innervation, and the well-known changes in AQueous Non-Radioactive cell solution (Promega cooperation, innervation associated with scar [14–16], we hypothesized Madison, USA) was added into each well including the control the absence of these ligands would result in decreased wells and incubated for 3h. Absorbance was read at 492nm innervation in scar. We also hypothesized that Ephrin using a Multiskan Transmit RC program (Labsystems, Frank- A2 and/or A5 signaling may affect healing through modula- land, Massachusetts, USA). The mean absorbance of all the À/À tion of keratinocyte migration. Here, using Ephrin A2 , samples in each genotype at each time pointwas calculated and À/À À/À À/À Ephrin A5 and Ephrin A2 A5 transgenic mice and an plotted to provide a measure of proliferation/viability of each excisional injury model, we have investigated the potential cell type over time. impact of Ephrin A2 and A5 on wound repair both in vitro and in vivo. 2.4. Keratinocyte isolation and culture Fur was removed from the dorsal mouse skin and cleaned with 2. Materials and methods povidone-iodine solution (Pfizer, Perth pty limited) and 70% ethyl alcohol using autoclaved cotton wool. Dorsal skin was 2.1. Animals harvested using scissors. Skin was collected in 1ml of TM Epigrow human epidermal keratinocyte complete media À/À À/À C57BL/6 wild type mice, Ephrin-A2 mice, Ephrin-A5 mice (Merck, Millipore, Massachusetts, USA). Skin tissue was cut À/À 2 and Ephrin-A2A5 mice were maintained in standard into pieces of 3–5mm . The pieces of skin were collected in 5ml TM housing with food and water provided ad libitum. Approval of Epigrow human epidermal keratinocyte complete media 1 TM was obtained by Institutional ethics committees and experi- with 20ml of fungizone (Gibco , Invitrogen , Carlsbad, 1 ments performed in accordance with the National Health and California, USA) and 100ml of kanamycin (Gibco , Invitro- TM Medical Research Council (NHMRC) Australian Code of Practice gen , Carlsbad, California, USA). 0.0127g of Dispase (Life TM for the Care and Use of Animals for Scientific Purposes. (RA/3/ technologies , Carlsbad, California, USA) per 5ml (2.4U/ml) 100/1162). was added. This solution was incubated for 24h at 4 C. 684 b u r n s 4 5 ( 2 0 1 9 ) 6 8 2 – 6 9 0 After 24h, the epidermis was separated from the dermis intramuscular) immediately prior to surgery. Oral paracetamol using sterile fine tip forceps and the pieces of epidermis in drinking water (1mg/ml) was given for 5days after injury. 1 TM transferred into 500ml of Tryple Select (Gibco , Invitogen , Once recovered the animals were group housed and allowed Carlsbad, California, USA) placed in a sterile petri dish. The food and water ad libitum. epidermal tissue pieces were spread out flat on the surface of Tryple Select and to prevent evaporation of the solution 2.7.
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