Species-specific regulation of angiogenesis by glucocorticoids reveals contrasting effects on inflammatory and angiogenic pathways

RESEARCH ARTICLE Species-specific regulation of angiogenesis by glucocorticoids reveals contrasting effects on inflammatory and angiogenic pathways Ruth Morgan1,2*, John Keen2, Daniel Halligan3, Alan O’Callaghan3, Ruth Andrew1, Dawn Livingstone1, Amber Abernethie1, Giorgia Maltese1, Brian Walker1, Patrick Hadoke1 a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 OPEN ACCESS Citation: Morgan R, Keen J, Halligan D, O’Callaghan A, Andrew R, Livingstone D, et al. (2018) Species-specific regulation of angiogenesis by glucocorticoids reveals contrasting effects on inflammatory and angiogenic pathways. PLoS ONE 13(2): e0192746. https://doi.org/10.1371/journal. pone.0192746 Editor: Christina L Addison, Ottawa Hospital Research Institute, CANADA Received: September 13, 2017 Accepted: January 30, 2018 Published: February 15, 2018 Copyright: © 2018 Morgan 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. 1 University/ BHF Centre for Cardiovascular Science, The Queen’s Medical Research Institute, University of Edinburgh, Edinburgh, United Kingdom, 2 Royal (Dick) School of Veterinary Studies, University of Edinburgh, Edinburgh, United Kingdom, 3 Fios Genomics Ltd, Nine Edinburgh Bioquarter, Edinburgh, United Kingdom * Abstract Glucocorticoids are potent inhibitors of angiogenesis in the rodent in vivo and in vitro but the mechanism by which this occurs has not been determined. Administration of glucocorticoids is used to treat a number of conditions in horses but the angiogenic response of equine vessels to glucocorticoids and, therefore, the potential role of glucocorticoids in pathogenesis and treatment of equine disease, is unknown. This study addressed the hypothesis that glucocorticoids would be angiostatic both in equine and murine blood vessels.The mouse aortic ring model of angiogenesis was adapted to assess the effects of cortisol in equine vessels. Vessel rings were cultured under basal conditions or exposed to: foetal bovine serum (FBS; 3%); cortisol (600 nM), cortisol (600nM) plus FBS (3%), cortisol (600nM) plus either the glucocorticoid receptor antagonist RU486 or the mineralocorticoid receptor antagonist spironolactone. In murine aortae cortisol inhibited and FBS stimulated new vessel growth. In contrast, in equine blood vessels FBS alone had no effect but cortisol alone, or in combination with FBS, dramatically increased new vessel growth compared with controls. This effect was blocked by glucocorticoid receptor antagonism but not by mineralocorticoid antagonism. The transcriptomes of murine and equine angiogenesis demonstrated cortisolinduced down-regulation of inflammatory pathways in both species but up-regulation of proangiogenic pathways selectively in the horse. Genes up-regulated in the horse and downregulated in mice were associated with the extracellular matrix. These data call into question our understanding of glucocorticoids as angiostatic in every species and may be of clinical relevance in the horse. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by the Biological and Biotechnological Sciences Research Council and Zoetis Animal Health (Grant No R42126 and R82976, author RAM,) with additional funding from PetPlan Insurance (S13-62). Work was carried out at the British Heart Foundation Centre of Research Excellence at the University of Introduction Angiogenesis, the formation of new blood vessels from existing vasculature, is essential for tissue repair [1]. Aberrant angiogenesis is an important feature of several disease processes including the growth of tumours [2], diabetic retinopathy [3] and rheumatoid arthritis [4]. PLOS ONE | https://doi.org/10.1371/journal.pone.0192746 February 15, 2018 1 / 18 Cortisol and angiogenesis Edinburgh. Authors DH and AO are employed by FIOS Genomics Ltd. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript and only provided financial support in the form of authors’ salaries and/or research materials. Competing interests: Authors DH and AO are employed by FIOS Genomics Ltd. This does not alter our adherence to PLOS ONE policies on sharing data and materials. The remaining authors have declared that no competing interests exist. Glucocorticoids at supra-physiological levels and in the presence of heparin, are potent inhibitors of angiogenesis in the chick embryo and rabbit corneal models [5]. At physiological concentrations, glucocorticoids inhibit angiogenesis in rodent models, both in vitro and in vivo [6]. When first described, this angiostatic effect presented a potentially significant therapeutic breakthrough in the prevention of tumour metastasis and aberrant angiogenesis [5, 7]. In addition, reduced angiogenesis is described in circumstances of chronic exposure to excess endogenous or exogenous glucocorticoids [8–10]. There has, however, been limited use of glucocorticoids as angiogenesis inhibitors in human medicine [11–13]. Glucocorticoids are frequently administered to veterinary species such as horses, in which prednisolone and dexamethasone are commonly prescribed for allergic dermatological and respiratory conditions. In horses glucocorticoids are also used for the initial treatment of tumours such as lymphoma but with limited success [14]. Glucocorticoid administration, and dysregulation of glucocorticoids in Equine Cushing’s Disease, have been implicated in the development of the vascular condition of the hoof, laminitis [15–17]. In chronic laminitis the blood vessels of the hoof fail to regenerate and there is evidence of a blunted angiogenic response with attenuation of the blood vessels and filling defects [18, 19]. The angiogenic response of equine vessels to glucocorticoids and, therefore, the potential role of glucocorticoids in pathogenesis and treatment of equine disease is unknown. The angiostatic effect of glucocorticoids is mediated by the glucocorticoid receptor in rodents [6] and in human endothelial cells [20] but the target cell and mechanism is unclear. Shikatani et al. found that corticosterone-treated rat endothelial cells exhibited reduced migration, through reduced RhoA and MMP-2 mediated proteolysis [21]. Migration of rat vascular smooth muscle cells and their MMP2 activity is also inhibited by dexamethasone but this effect is not observed in human smooth muscle cells [22]. Glucocorticoids prevent tube-like structure formation by human endothelial cells [20]. Logie et al. demonstrated that cortisol induces cytoskeletal disruption, interfering with cell-to-cell contact of endothelial cells, but does not inhibit their proliferation or migration [20]. Analysis of a selection of candidate genes in that study showed (...truncated)