Thyroxine (T4) may promote re-epithelialisation and angiogenesis in wounded human skin ex vivo

RESEARCH ARTICLE Thyroxine (T4) may promote re-epithelialisation and angiogenesis in wounded human skin ex vivo Guo-You Zhang1☯, Ewan A. Langan2,3☯, Natalia T. Meier4, Wolfgang Funk5, Frank Siemers6, Ralf Paus7,8* a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 1 Department of Plastic and Reconstructive Surgery, Shanghai Ninth People’s Hospital, Shanghai Jiaotong University School of Medicine, Shanghai, China, 2 Department of Dermatology, University of Lübeck, Lübeck, Germany, 3 Centre for Dermatology Research, University of Manchester, and NIHR Manchester Biomedical Research Centre, Manchester, United Kingdom, 4 Department of Pathology, University of Lübeck, Lübeck, Germany, 5 Klinik Dr. Funk, Munich, Germany, 6 Department of Plastic and Hand Surgery, BG Klinikum Bergmannstrost, Halle/Salle, Germany, 7 Department of Dermatology & Cutaneous Surgery, University of Miami Miller School of Medicine, Miami, Florida, United States of America, 8 Centre for Dermatology Research, University of Manchester, and NIHR Manchester Biomedical Research Centre, Manchester, United Kingdom ☯ These authors contributed equally to this work. * OPEN ACCESS Citation: Zhang G-Y, Langan EA, Meier NT, Funk W, Siemers F, Paus R (2019) Thyroxine (T4) may promote re-epithelialisation and angiogenesis in wounded human skin ex vivo. PLoS ONE 14(3): e0212659. https://doi.org/10.1371/journal. pone.0212659 Editor: Vincenzo Miragliotta, University of Pisa, ITALY Received: October 11, 2017 Accepted: February 7, 2019 Published: March 29, 2019 Copyright: © 2019 Zhang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the manuscript and its Supporting Information files. Abstract There is a pressing need for improved preclinical model systems in which to study human skin wound healing. Here, we report the development and application of a serum-free full thickness human skin wound healing model. Not only can re-epithelialization (epidermal repair) and angiogenesis be studied in this simple and instructive model, but the model can also be used to identify clinically relevant wound-healing promoting agents, and to dissect underlying candidate mechanisms of action in the target tissue. We present preliminary ex vivo data to suggest that Thyroxine (T4), which reportedly promotes skin wound healing in rodents in vivo, may promote key features of human skin wound healing. Namely, T4 stimulates re-epithelialisation and angiogenesis, and modulates both wound healing-associated epidermal keratin expression and energy metabolism in experimentally wound human skin. Functionally, the wound healing-promoting effects of T4 are at least partially mediated via fibroblast growth factor/fibroblast growth factor receptor-mediated signalling, since they could be significantly antagonized by bFGF-neutralizing antibody. Thus, this pragmatic, easy-to-use full-thickness human skin wound healing model provides a useful preclinical research tool in the search for clinically relevant candidate wound healing-promoting agents. These ex vivo data encourage further pre-clinical testing of topical T4 as a cost-efficient, novel agent in the management of chronic human skin wounds. Funding: This work was supported in part by a grant from the Federal Ministry of Economics, Germany (AiF/ZIM Programme) to R.P. G-Y Zhang was supported by 2011 Chinese government award for outstanding self-financed students abroad (China Scholarship Council) and National Natural Science Foundation of China (81101430). PLOS ONE | https://doi.org/10.1371/journal.pone.0212659 March 29, 2019 1 / 14 Topical Thyroxine may promote human skin wound healing Competing interests: The authors have declared that no competing interests exist. Abbreviations: bFGF, basic fibroblast growth factor; CK, cytokeratin; DAPI, 4’-6-diamidino-2phenylindole; ET, epithelial tongue; FGFR1, fibroblast growth factor receptor 1; HF, hair follicle; IR, immunoreactivity; T4, thyroxine; TH, thyroid hormones; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labelling. Introduction Retarded healing of human skin wounds, which may result in ulceration, represents an increasing, global healthcare and quality-of-life challenge, especially in the context of an aging population [1–5]. Indeed, there is a pressing need for the identification of effective, safe and cost-efficient wound healing promoters which can be introduced into clinical practice [6]. In order to meet this need, it is critical to have simple and pragmatic, predictive model systems in which new candidate promoters of human skin wound healing can be instructively studied at the preclinical level [7, 8]. Though modelling wound healing in so-called 3D skin “equivalent” culture systems is informative [9], such systems usually lack skin appendages, immune cells, for example macrophages and mast cells, and other resident skin cells, and do not reflect the tissue tension characteristics of human skin, all of which are already known to significantly modulate cutaneous would healing [10–16]. Therefore, we and others have advocated the use of experimentally wounded full-thickness human skin ex vivo, ideally under defined, serumfree organ culture conditions, as well as the systematic testing of agents that have already been licensed for clinical use [7]. Thyroid hormones (THs) are of special interest in this context, since human skin and hair follicles are classical TH target organs [17–27], while thyroid diseases affect skin structure and function on multiple levels [28–30]. For example, L-thyroxine (T4) promotes human hair growth [19] and stimulates wound healing in vivo in rats [31] and mice [32]. Moreover, T4 operates as the chief endocrine control of amphibian metamorphosis [33], suggesting that it can act as a powerful morphogen. In addition, T4 is one of the most frequently administered hormones in clinical medicine, where it has been in extensive use for decades, its toxicology is very wellexamined [34], and it is relatively inexpensive. Yet, the potential clinical utility of T4 in a dermatological setting, namely as a candidate wound healing promoter, is yet to be fully explored [30]. Given the reported wound healing-promoting properties of T4 in rodents [31, 32] and the strong interdependence of cutaneous wound healing, hair follicle (HF) cycling, HF neogenesis and skin stem cell activities [35–37], we hypothesized that T4 may also promote human skin wound healing. In order to probe whether T4 directly impacted on human skin wound healing, i.e. in the absence of other systemic/endocrine inputs, we tested our hypothesis in serumfree organ culture of full-thickness human skin [38] that had been experimentally wounded, using a “punch-in-a-punch” design [7, 39]. (...truncated)