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The Fifth Epidermal Growth Factor-Like Region of Thrombomodulin Exerts Cytoprotective Function and Prevents SOS in a Murine Mode

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The Fifth Epidermal Growth Factor-Like Region of Thrombomodulin Exerts Cytoprotective Function and Prevents SOS in a Murine Mode Bone Marrow Transplantation (2017) 52, 73–79 © 2017 Macmillan Publishers Limited, part of Springer Nature. All rights reserved 0268-3369/17 www.nature.com/bmt ORIGINAL ARTICLE The fifth epidermal growth factor-like region of thrombomodulin exerts cytoprotective function and prevents SOS in a murine model T Ikezoe1,2, J Yang1,7, C Nishioka1,BPan1,3,KXu3, M Furihata4, K Nakamura5, H Yurimoto5, Y Sakai5, G Honda6 and A Yokoyama1 The present study found that the fifth epidermal growth factor-like domain of thrombomodulin (TME5) possesses the cytoprotective function in association with an increase in levels of anti-apoptotic myeloid cell leukemia-1 protein in an activated protein C-independent manner in human umbilical vein endothelial cells (HUVECs). Importantly, TME5 counteracted calcineurin inhibitor-induced vascular permeability and successfully prevented monocrotaline-induced sinusoidal obstruction syndrome (SOS) in a murine model. Taken together, TME5 may be useful for preventing or treating lethal complications that develop after hematopoietic stem cell transplantation such as SOS and thrombotic microangiopathy in which endothelial cell damage has a role. Bone Marrow Transplantation (2017) 52, 73–79; doi:10.1038/bmt.2016.195; published online 18 July 2016 INTRODUCTION by TME45 and is independent of APC (Figure 1).7 Interestingly, Thrombomodulin (TM) is a glycosylated type I transmembrane TME45 also stimulates proliferation of human umbilical vein 7 molecule with multiple domains that include an NH2-terminal endothelial cells (HUVECs) and promotes angiogenesis in vivo. lectin-like region, six tandem epidermal growth factor (EGF)-like Recombinant human soluble TM (rTM) includes the active, structures, an O-glycosylation site-rich domain, a transmembrane extracellular domain of TM and inactivates coagulation. Use of rTM domain and a cytoplasmic tail domain (Figure 1).1 TM is for the treatment of disseminated intravascular coagulation was ubiquitously expressed on endothelial cells and binds to approved in Japan in 2008, and we have since rescued individuals thrombin, forming a 1:1 complex via the fourth and fifth regions with disseminated intravascular coagulation complicated by of an EGF-like domain (TME45) and acting as an anticoagulant.2 various underlying diseases, including transplantation-associated In addition, the thrombin–TM complex activates protein C to microangiopathy (TMA) and sinusoidal obstruction syndrome (SOS), as well as engraftment syndrome following hematopoietic produce activated protein C (APC), which inactivates factors – VIIIa and Va in the presence of protein S, thereby inhibiting further stem cell transplantation (HSCT).8 10 The pathogenesis of these thrombin formation.3 The minimum structure that is essential for potentially lethal complications is based on endothelial damage. generating APC is localized in TME456 repeats of the EGF-like Thus, they are referred to as endothelial syndromes.11 Use of rTM domain (Figure 1).4 Interestingly, TM possesses anti-inflammatory for treatment of coagulopathy that develops after HSCT properties; the lectin-like domain of TM binds to and inactivates significantly prolongs overall survival of patients compared to high-mobility group box 1 protein (HMGB1), a nuclear those with coagulopathy who did not receive rTM mainly because architectural chromatin-binding protein that stimulates rTM improves the survival of patients with endothelial syndrome production of inflammatory cytokines such as interleukin 6 (IL-6) that triggers coagulopathy at day 100 after HSCT.12 rTM appears to and TNF-α via toll-like receptor 4 and the receptor for be relatively safe, as a phase III clinical trial comparing the efficacy advanced glycation end products after being released from and safety between rTM and low-dose heparin showed that the necrotic cells or secreted from inflammatory cells such as incidence of bleeding-related adverse events up to 7 days activated macrophages.5 In addition, the lectin-like domain of after the start of infusion was lower in the rTM group than in TM inhibits lipopolysaccharide-induced cytokine production and the heparin group (43.1 vs 56.5%, P = 0.0487), although this adhesion of neutrophils to endothelial cells in association with clinical trial did not include patients who received HSCT.13 suppression of extracellular signal-regulated kinase (ERK) and Notably, one patient with SOS after HSCT developed a fatal nuclear factor κB (NF-κB).6 Intriguingly, TM exerts endothelial intracranial hemorrhage during treatment with rTM.14 These cytoprotective effects against inflammatory cytokines and observations prompted us to develop a novel agent that does calcineurin inhibitors such as cyclosporine A (CsA) and tacrolimus not affect the coagulation system to safely prevent or treat (FK506) via upregulation of myeloid cell leukemia sequence 1 endothelial syndrome after HSCT. (Mcl-1) proteins in a process that is mediated by the ERK signal The present study attempted to identify the minimum structure transduction pathway.7 This cytoprotective effect is also mediated of TME45 that is responsible for the cytoprotective function of TM 1Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan; 2Department of Hematology, Fukushima Medical University, Fukushima City, Fukushima, Japan; 3Department of Hematology, The Affiliated Hospital of Xuzhou Medical College, Xuzhou, China; 4Department of Pathology, Kochi Medical School, Kochi University, Nankoku, Kochi, Japan; 5Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto, Japan and 6Medial Affairs Department, Asahi Kasei Pharma, Kanda Jinbocho, Chiyoda-ku, Tokyo, Japan. Correspondence: Dr T Ikezoe, Department of Hematology, Fukushima Medical University, Hikarigaoka 1, Fukushima City, Fukushima 960-1295, Japan. 7Current address: J Yang, Jiangsu Center for the Collaboration and Innovation of Cancer Biotherapy, Cancer Institute, Xuzhou Medical College, Xuzhou, Jiangsu 221002, China. E-mail: [email protected] Received 13 April 2016; revised 6 June 2016; accepted 9 June 2016; published online 18 July 2016 TME5 prevents SOS T Ikezoe et al 74 Lectin-like domain Anti-inflammatory effect E1 E2 E3 EGF-like domain E4 Anti-coagulation effect E5 Cytoprotective effect E6 O-glycosylation site-rich domain Transmembrane domain Endothelial cell Cytoplasmic tail domain Figure 1. The structure and function of TM. E1–6, six repeats of EGF-like domain of TM. A full color version of this figure is available at the Bone Marrow Transplantation journal online. and explored the potential of polypeptides comprising this Blocking antibodies structure as a cytoprotective agent in vitro and in vivo. Antibodies from the TM-specific antibody clone RTM96 (Hycult Biotech Inc., Plymouth Meeting, PA, USA) and clone Phx-01 (Ab24595, Abcam, Cambridge, MA, USA) were used as blocking antibodies to the fourth MATERIALS AND METHODS and fifth region of the EGF-like domain of TM, respectively. Cells HUVECs were purchased from Lonza Walkersville Inc. (Basel, Switzerland) Western blot analysis and cultured in EBM-2 culture medium supplemented with EGM-2 Western blot analysis was performed as described previously.17 The endothelial cell growth factors (Lonza Walkersville Inc.). Human endothelial membrane was probed sequentially with the antibodies. Antibodies EA.hy926 cells were purchased from ATCC (Manassas, VA, USA) and specific to p-ERK (T202/Y204) (Cell Signaling Technology, Danvers, MA, cultured with DMEM (Sigma-Aldrich, St Louis, MO, USA). USA, 9101, 1:1000), ERK (Cell Signaling Technology, 9102, 1:1000), Mcl-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-819, 1:1000) and Reagents GAPDH (Abcam, 1:1000) were used. Band intensities were measured by ImageJ software (Wayne Rasband, NIH). rTM was provided by Asashi Kasei Pharma (Tokyo, Japan). CsA, FK506, monocrotaline (MCT) and IL-1β were purchased from Sigma-Aldrich. RNA isolation and reverse transcriptase-PCR Generation of TME5 To quantify the expression of Mcl-1, E-selectin and intracellular adhesion fi molecule 1 (ICAM1), real-time reverse transcriptase-PCR (RT-PCR) was The fth EGF-like domain of TM (TME5) was synthesized and cloned into employed as described previously.18 We measured the expression of 18S the pcDNA3.1/V5-His-A expression vector (Invitrogen, Carlsbad, CA, USA) α for normalization. The primer sequence information for Mcl-1 and 18S and pPICZ A expression vector (Invitrogen). These vectors were was described elsewhere.7 The primers for PCR to detect E-selectin transfected into either COS-1 cells or Pichia pastoris strain SMD1163 (his4 15 (5′-AGCTACCCATGGAACACGAC-3′ and 5′-ACGCAAGTTCTCCAGCTGTT-3′) pep4 prb1), respectively. For Pichia expression, the DNA coding region for and ICAM1 (5′-TTCACACTGAATGCCAGCTC-3′ and 5′-GTCTGCTGAGACCCC TME5 was synthesized according to the preferred codon usage of the TCTTG-3′) were synthesized by Sigma-Aldrich Japan (Tokyo, Japan). methylotrophic yeasts16 and the transformant cells were grown to high- cell density on methanol medium.16 The generated TME5 proteins were purified with a His-tagged protein purification kit (MBL, Aichi, Japan) or Ni Vascular permeability assay in vitro Sepharose 6 Fast Flow (GE Healthcare, Little Chalfont, UK). The TME5 Effects of test agents on vascular permeability were measured with a vascular polypeptide was synthesized by Peptide Institute Inc. (Osaka, Japan). permeability assay kit (Millipore, Billerica, MA, USA) as described previously.7 Plasmids and
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