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

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

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 production of proteins Apoptosis assay D123 comprising the extracellular domain of TM and D1 comprising the The ability of a calcineurin inhibitor to induce apoptosis in HUVECs was lectin-like domain of TM and TME45 were cloned into the FLAG-TEV–tagged measured using the annexin V-fluorescein isothiocyanate apoptosis detec- pcDNA3.1/V5-His-TOPO expression vector (Invitrogen). COS-1 cells were tion kit (Pharmingen, Inc., San Diego, CA, USA) as previously described.7 transfected with each vector, and His-tagged mutant TM proteins were purified using the His-tagged protein purification kit (MBL) as described previously.7 GPR15 cDNA was purchased from the Mammalian Gene Protein C activation assay Collection (BC069437) and amplified by PCR. The product was cloned into The ability of TM to accelerate thrombin-catalyzed protein C activation was the pLenti6.3/V5-TOPO vector (Invitrogen) and transduced into HUVECs. measured as previously described.19 APC hydrolytic activity of S-2366 was monitored at 405 nm. One unit of TM activity was defined as 1 μmol of p-nitroaniline formed/min. Proliferation assays HUVECs (5 × 103 cells) were cultured in a 96-well plate for 24 h in the presence of the indicated reagent. Bromodeoxyuridine (10 μM/well) Pharmacokinetics of TME5 in vivo was added and incubated for an additional 4 h. The quantity of TME5 (1 mg per kg body weight) was given to ICR mice (6-week-old female) bromodeoxyuridine incorporated into the newly synthesized DNA via IP injection. Blood collection was initiated 3 h after injection and was assessed in accordance with the manufacturer’s protocol (Roche, continued up to 72 h (3, 6, 12, 24, 48 and 72 h after injection). The collected Basel, Switzerland). blood samples were subjected to ELISA to monitor the plasma levels of TME5.

Bone Marrow Transplantation (2017) 73 – 79 © 2017 Macmillan Publishers Limited, part of Springer Nature. TME5 prevents SOS T Ikezoe et al 75 The PVC microtiter plate was coated with TM-specific antibody (Abcam, anti-apoptotic Mcl-1 protein in HUVECs. The blocking antibody ab6980). An HRP-conjugated TM-specific polyclonal antibody (Bioss, Woburn, for TME5 but not for TME4 hampered the ability of TME45 MA, USA, bs-0525R-HRP) was used as a secondary antibody. expressed in COS-1 cells to induce expression of Mcl-1 in HUVECs (Figure 2a), suggesting that the cytoprotective Murine in vivo endothelial cell damage model function of TME45 is preserved in TME5. We next expressed All animal care and experimental procedures were approved by the TME5 protein in COS-1 cells and compared its ability to induce Institutional Animal Care and Use Committee of Kochi University. C57BL6 Mcl-1 with that of D123 (whole extracellular domain of TM) and 7 mice (6-week-old female) were treated with either FK506 (5 mg per kg body TME45. Consistent with a previous study, TME45 (1.5 nM) more weight) or control diluent (saline) every day for 7 days by IP injection to potently induced Mcl-1 than D123 (1.5 nM) in HUVECs (Figure 2b). induce vascular cell damage as previously described.7 Either TME5 (0.5 mg The ability of TME5 to induce Mcl-1 was almost identical to per kg body weight) or control diluent (double distilled water) was given to that of TME45 when used at the equivalent molar concentration mice every other day by IP injection for 7 days. At the end of experiments, (Figure 2b). mice were killed, and blood was collected. Plasma was separated by centrifugation, and the plasma levels of murine TM, a marker of endothelial cell damage, and fibrin/fibrinogen degradation products (FDPs) were assessed TME5 blocks CsA-induced apoptosis and capillary leakage with a murine TM ELISA kit (Uscn Life Science, Tokyo, Japan) and a murine We next examined whether TME5 possesses anti-apoptotic effects FDP ELISA kit (Uscn Life Science), respectively, following the manufacturer’s in HUVECs. Following previous experiments,7 we utilized the instructions. At the same time, the kidneys and liver were removed from mice calcineurin inhibitor CsA to induce endothelial cell damage in and mashed to make a single-cell suspension. These cells were stained with HUVECs. CsA-induced apoptosis was significantly attenuated CD31-specific antibody (Biolegend; 1:1000), and positive cells were isolated by sorting using JSAN (Bay Bioscience, Ltd., Kobe, Japan). RNA was extracted and when HUVECs were cultured with CsA in the presence of TME5 subjected to real-time RT-PCR to measure the levels of murine Mcl-1. expressed in COS-1 cells; exposure of HUVECs to CsA alone (4 mg/ml) induced a mean of 30% of these cells to become annexin V positive. On the other hand, a mean of 12% of HUVECs SOS murine model became annexin V positive when these cells were exposed to the ICR mice (6-week-old females) were purchased from Japan SLC, Inc. same concentration of CsA in combination with TME5 expressed (Shizuoka, Japan). The treatment protocol followed the rat SOS model with β fi 20 fl in COS-1 cells (0.14 nM) (Figure 3a). In addition, CsA and IL-1 some modi cations. Brie y, MCT (200 mg per kg body weight) was fi administered to mice via IP injection on day 1 to induce SOS. Concurrently, induced capillary leakage, which was also signi cantly blocked by either TME5 (1 mg per kg body weight, n=6) or control diluent (double TME5 expressed in COS-1 cells in vitro (Figure 3b). distilled water, n=6) was administered to these mice via IP injection on days 1 and 2. Blood was withdrawn, and plasma levels of aspartate TME5 stimulates angiogenesis aminotransferase and alanine aminotransferase were measured on day 3 As rTM stimulates angiogenesis,7 we explored whether of the experiments. After the blood collection, mice were killed, and the fi proangiogenic activity is preserved in TME5. Following a previous ascites volume was determined. Organs were removed, and paraf n- 21 embedded sections were made, stained with hematoxylin and eosin and study, we utilized P. pastoris to generate TME5 protein in subjected to pathological examination. large quantities to perform in vivo experiments. Also, we synthesized TME5 polypeptide comprising 40 amino acids and compared their proangiogenic activity in vitro. The synthesized Statistical analysis TME5 appeared to possess more potent activity for promoting Statistical analysis was performed to assess the difference between two angiogenesis than Pichia- or COS-1-expressed TME5 or rTM groups under multiple conditions using one-way ANOVA followed by fi Bonferroni multiple comparison tests using PRISM statistical analysis ( gure not shown). We therefore decided to utilize synthesized software (GraphPad Software, La Jolla, CA, USA). TME5 for further in vivo experiments.

Anticoagulant activity and pharmacokinetics of TME5 RESULTS TME45 and TME456 bind to thrombin and activate protein C, The ability of TM to induce Mcl-1 is preserved in TME5 respectively.2,4 We thus assessed whether TME5 was able to We ﬁrst utilized a blocking antibody for either TME4 or TME5 generate APC. COS-1 cell-expressed TME45 retained detectable to determine which structure is responsible for induction of levels of activity to generate APC, although its ability was o1% in

a b Control D123 TME45 TME5 TME45 - ++ + Anti-TME5 ab - - - + Mcl-1 Anti-TME4 ab - - + - GAPDH Mcl-1 10 8

GAPDH 6

2 Intensity of Mcl-1 0

(fold induction of control) Control D123 TME45 TME5 Figure 2. TME5 increases Mcl-1 in HUVECs. (a) Western blots. HUVECs were pre-incubated with a blocking antibody (20 mg/mL) for either TME4 (clone RTM96) or TME5 (clone Phx-01). Ten minutes later, HUVECS were exposed to TME45. Forty-eight hours later, proteins were extracted and subjected to western blot analyses. (b) Western blots. HUVECs were cultured in the presence of control diluent, D123, TME45, or TME5 protein at an equivalent molar concentration (1.5 nM). After 48 h, proteins were extracted and subjected to western blot analyses. The membrane was sequentially probed with the indicated antibodies (upper panel). The experiments were repeated three times, and band intensity was quantiﬁed with ImageJ software. Results are summarized as a bar graph (lower panel). Error bars show s.d.

© 2017 Macmillan Publishers Limited, part of Springer Nature. Bone Marrow Transplantation (2017) 73 – 79 TME5 prevents SOS T Ikezoe et al 76 a 50 Late apoptosis 40 Early apoptosis 30 * P <0.05 20 * * 10 Apoptotic cells(%) 0 CsA (mg ml-1) - 2 4 - 24 TME5 (0.14 nM) --- + ++

b * 8 * 3

6 2 4 RFU RFU 1 2 (fold of control) (fold of control) 0 0 CsA (4 mg ml-1) - + --+ IL-1β (100 ng ml-1) + - + TME5 (0.14 nM)--+ + TME5 (0.14 nM) --+ + Figure 3. TME5 blocks CsA-induced apoptosis and vascular permeability. (a) Apoptosis assay. HUVECs were exposed to TME5 and/or CsA. After 48 h, cells were harvested, stained with anti-annexin V antibody and propidium iodide (PI), and subjected to fluorescence-activated cell sorting to quantify the population of cells undergoing apoptosis. Early and late apoptosis indicates the annexin V-positive and PI-negative or annexin V-positive and PI-positive populations, respectively. Experiments were performed three times in a triplicate culture plate. Error bars show s.d. Statistical significance was assessed by one-way ANOVA followed by Bonferroni multiple comparison tests. (b) Vascular permeability assay. AN HUVEC monolayer was exposed to either CsA+/ − TME5 (left panel) or IL-1β+/ − TME5 (right panel). After 12 h, FITC-Dextran was added to the culture medium. The extent of vascular permeability was quantified by measuring the fluorescence of the bottom plate solution affected by permeated FITC-Dextran. Results represent fold of the control well. Data represent means ± s.d. of three independent experiments performed in duplicate cultures. Statistical significance was assessed by one-way ANOVA followed by Bonferroni multiple comparison tests. RFU, relative fluorescent unit.

ab 10000 ) 1000000 409600 -1

100000 8000 ) 10000 -1 6000 1758.4 1000 4000 100 TME5 (ng ml

2000 10 0

Anticoagulant activity (unit mol 1 0 rTM TME45 TME5 03612244872 Hrs Figure 4. (a) TME5 does not generate APC. rTM, TM45 or TME5 was incubated with protein C and thrombin in Eppendorf tubes to generate APC. The amount of APC generated was measured by monitoring the hydrolysis of the chromogenic substrate, S-2366, at 405 nm. The bar graph represents one of the two experiments performed independently. (b) Pharmacokinetics of synthesized TME5 in ICR mice. Six-week-old female ICR mice (n=2) received synthesized TME5 (1 mg per kg body weight) via IP injection. Blood samples were sequentially withdrawn from the retro-orbital venous plexus at the indicated time point, and plasma levels of TME5 were measured by ELISA. Results represent one of the two experiments performed independently. The blue and green lines indicate two different mice. A full color version of this ﬁgure is available at the Bone Marrow Transplantation journal online.

comparison with rTM. Importantly, COS-1 cell-expressed TME5 TME5 prevents tacrolimus (FK506)-induced endothelial injury completely lost the ability to generate APC (Figure 4a), conﬁrming in vivo that TME5 does not affect the anticoagulation system. To We examined the cytoprotective effect of synthesized TME5 in a determine the dosing schedule in in vivo experiments, we FK506-induced endothelial injury murine model. As shown monitored the plasma levels of TME5 after IP injection of previously,7 administration of FK506 for 7 days caused endothelial 1 mg/kg synthesized TME5 into ICR mice. The plasma levels injury as evidenced by an increase in the plasma levels of TM in of TME5 increased steeply and reached a maximum concentration of ~ 8000 ng/mL at 12 h and decreased to basal levels by 72 h with association with a slight decrease in levels of Mcl-1 in vascular a half-life of 24 h (Figure 4b). From these observations, we decided endothelial cells in the kidney and liver (Figures 5a and b). Plasma to give synthesized TME5 to mice every another day in in vivo levels of FDPs were also increased (Figure 5b), suggesting that the experiments. coagulopathy resulted from endothelial injury. Administration of

a Kidney Liver 15 15 * N.S ** *

10 10

N.S

5 5 N.S (fold of control) (fold of control) Relative expression of MCL-1 0 Relative expression of MCL-1 0 - FK TME5 FK+TME5 - FK TME5 FK+TME5

** b ** 50 N.S 60 ) -1 40 )

-1 40 30

20 20 FDP (ng ml 10 Thrombomodulin (ng ml 0 0 -FKTME5 FK+TME5 -FKTME5 FK+TME5 Figure 5. TME5 protects endothelial cells from damage caused by FK506 in vivo.(a) Real-time RT-PCR. C57BL6 mice (n=3) were treated with either FK506 (5 mg per kg body weight) or control diluent every day for 7 days by IP injection. Either synthesized TME5 (0.5 mg per kg body weight) or control diluent was given to mice every other day by IP injection for 7 days. At the end of the experiments, mice were killed, and kidneys and livers were removed from mice and mashed to make a single-cell suspension. These cells were stained with an anti-CD31 antibody, and positive cells were isolated by sorting. RNA was extracted and subjected to real-time RT-PCR to measure the levels of murine Mcl-1. Transcripts of TM were normalized according to the amount of 18S transcript. Results represent fold of the control well without agents by one-way ANOVA followed by Bonferroni multiple comparison tests. (b) ELISA. At the end of the experiments, blood was collected, and plasma was separated by centrifugation. The plasma levels of murine TM and FDPs were assessed by murine TM and FDP ELISA kits, respectively. Statistical analysis was performed by one-way ANOVA followed by Bonferroni multiple comparison tests. **Po0.01. Data in (a) are means from three independent experiments. Data in (b) represent the results of three experiments performed independently; error bars show s.e.m. NS, not statistically significant; FK, FK506. synthesized TME5 alone markedly increased levels of Mcl-1, and lectin-like domain of TM (D1) known to inhibit ERK/Mcl-1 when FK506 was administered to mice together with synthesized pathway7 was not able to counteract MCT-induced ascites TME5, FK506-induced downregulation of Mcl-1 in endothelial cells retention and elevation of the liver enzymes (data not shown), was completely abolished (Figure 5a). The FK506-induced increase suggesting the specificity of TME5 against SOS. in plasma levels of TM and FDPs was also hampered when mice received FK506 in combination with synthesized TME5 (Figure 5b). DISCUSSION 11 TME5 prevents MCT-induced SOS in vivo Endothelial syndrome is a potentially lethal complication after HSCT. Our previous clinical observations showed that the Pyrrolizidine alkaloid MCT (200 mg per kg body weight) was given use of rTM successfully rescues individuals with endothelial to ICR mice to induce SOS. A dark reddish color, a sign of 8–10 fi congestion, was noted on the surface of the liver that was syndrome and signi cantly prolongs overall survival of transplant patients with coagulopathy complicated by endothelial removed from MCT-treated mice 72 h after beginning the 12 experiments (Figure 6a). Massive ascites was also noted in syndrome. These observations indicate the cytoprotective roles MCT-treated mice in association with a marked increase in plasma of rTM in the context of HSCT. Indeed, a recent in vitro study found that TME45 protects endothelial cells from cytokines and levels of the liver enzymes aspartate aminotransferase and alanine 7 aminotransferase (Figures 6b and c). Real-time RT-PCR showed calcineurin inhibitor-induced apoptosis, supporting the clinical fi that levels of E-selectin and ICAM1, a known endothelial insult bene t of rTM for transplant patients. The present study 22 fi marker that is increased in patients who develop SOS after HSCT, attempted to de ne the minimal region of TME45 with were elevated in livers isolated from MCT-treated mice compared cytoprotective activity and identified TME5 as a minimum with those in control diluent-treated mice (Figure 6d). In addition, structure (Figure 2). Intriguingly, the use of TME5 significantly pathological examination showed destructive changes of the attenuated FK506- and MCT-induced endothelial damage and liver sinusoid accompanying hemorrhagic necrosis in zone 3 SOS, respectively, in a murine model (Figures 5 and 6). (pericentral zone) and zone 2 (transition zone). Normal structure of FK506-induced endothelial cell damage is one type of pathogen- central vein was not detectable in zone 3, and sinusoidal dilatation esis associated with the development of TMA. We therefore was noted in zone 1 (periportal zone). Collectively, these assume that the use of TME5 may be useful for preventing or observations suggested the development of SOS in MCT-treated treating TMA. The limitation of this study is the use of MCT to mice (Figure 6e). Importantly, when MCT was given to mice induce SOS in vivo, which is not clinically relevant. Future together with synthesized TME5, all of these hepatotoxic changes experiments will evaluate the cytoprotective effects of TME5 in a were alleviated (Figures 6a–e), indicating the prophylactic roles of recently reported SOS model induced by cyclophosphamide and TME5 against MCT-mediated SOS. On the other hand, the busulfan followed by HSCT.23

Contol MCT MCT+TME5 μ ** 2000

1500

1000

500

Abdominal dropsy ( 0 - MCT MCT+TME5

c AST ALT d E-selectin ICAM ** ** 10.0 5000 7500 7. 5 ** ** ** ** ** * 4000 7. 5 5000 5.0 3000 5.0 IU/L IU/L 2000 2500 2.5 2.5 1000

0 0 0.0

0.0 induction of control Fold Control MCT MCT+TME5 Control MCT MCT+TME5 induction of control Fold Control MCT MCT+TME5 Control MCT MCT+TME5

e Control MCT MCT+TME5

PT PT

Figure 6. TME5 prevents development of MCT-induced SOS in vivo.(a) Macroscopic examination. Six-week-old female ICR mice (n=6 per group) were treated with either control diluent or MCT (200 mg per kg body weight) by IP injection. Either control diluent or synthesized TME5 (1 mg per kg body weight) was given to mice on days 1 and 2 by IP injection. On day 3 of the experiment, mice were killed, and livers were photographed. The pictures show representative results of experiments performed three times independently. (b) Volume of ascites. After euthanasia, ascites was harvested from mice (n=6 per group) and weighed. (c) Plasma levels of liver enzymes. After euthanasia, blood was collected from mice (n=6 per group), and plasma levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. (d) Real-time RT-PCR. Livers were also removed after euthanasia, and RNA was extracted and subjected to real-time RT-PCR. (e) Pathological examination of the liver. At the end of the experiments, livers were removed from mice (n=6 per group) and stained with hematoxylin and eosin (HE). The pictures show the representative view of HE staining. PT, portal tract; CV, central vein. Data in (b–d) are means from three independent experiments; error bars show s.e.m. Statistical analysis was performed by one-way ANOVA followed by Bonferroni multiple comparison tests. *Po0.05, **Po0.01.

rTM is a potent and relatively safe newly developed Expression levels of the adhesion molecule ICAM1, which is anticoagulant. However, we have to be very cautious about regulated, at least in part, by HMGB1, were significantly lower in hemorrhages when we administer rTM to transplant patients with livers removed from rTM-treated rats than in those from normal endothelial syndrome because endothelial syndrome is frequently saline-treated rats. In parallel, fewer neutrophils were noted in 24 complicated with transfusion-refractory thrombocytopenia. livers removed from rTM-treated rats than normal saline-treated Importantly, TME5 does not affect the coagulation pathway, as it rats.20 The lectin-like domain of TM binds to and degrades does not generate APC (Figure 4a). TME5 may be a suitable HMGB1.5 In the present experiments, we utilized TME5 lacking the cytoprotective agent for transplant patients with a high risk of lectin-like domain of TM. The cytoprotective effects mediated by hemorrhage. Another advantage of TME5 over rTM relates to ERK; TME5 were powerful enough to alleviate MCT-induced SOS. TME5 exerts its cytoprotective function via activation of ERK. On TM mitigates radiation-induced bone marrow failure via the other hand, the lectin-like domain of rTM inactivates ERK,6 APC-dependent mechanisms in a murine model.27 The authors which may blunt the cytoprotective function of rTM. Other investigators recently showed that a combination of IV assumed that the TM/APC system protected the bone marrow and SC injection of rTM effectively blocks the development of microenvironment, the so-called niche, from radiation injury and MCT-induced SOS in rats and significantly prolongs their survival supported hematopoiesis. TME5 could also contribute to cytopro- compared with normal saline-treated rats.20 They demonstrated tection of the bone marrow niche in an APC-independent manner. that administration of MCT to rats increases serum levels of The cell surface molecules that mediate proangiogenic effects HMGB1 and non-histone nuclear proteins, and that rTM effectively of TM had remained unknown for a long time. Of note, fibroblast counteracts the MCT-mediated increase in levels of HMGB1. growth factor receptor has been recently identified as a candidate HMGB1 is released from necrotic cells as well as activated receptor for TM.28 Future experiments will explore whether TME5 inflammatory cells and stimulates further production of inflam- exerts the cytoprotective function via fibroblast growth factor matory cytokines in association with activation of NF-κB.25,26 receptor.

Bone Marrow Transplantation (2017) 73 – 79 © 2017 Macmillan Publishers Limited, part of Springer Nature. TME5 prevents SOS T Ikezoe et al 79 The maximal plasma concentration of TM in the individuals who with recombinant human soluble thrombomodulin. Bone Marrow Transplant received a single dose of rTM 380 U/kg (equivalent to 0.06 mg/kg) 2010; 45:783–785. 29 was 1495.4 ng/ml (~233 nM) with a half-life of 28 h. We 10 Ikezoe T, Takeuchi A, Taniguchi A, Togitani K, Yokoyama A. Recombinant human soluble thrombomodulin counteracts capillary leakage associated with M previously showed that low dose of rTM (5-50 n ) produced 46 – cytoprotective activity.7 The present study also found that sub- engraftment syndrome. Bone Marrow Transplant 2011; : 616 618. 11 Carreras E, Diaz-Ricart M. The role of the endothelium in the short-term complications nanomolar concentrations of TME5 effectively protected endothe- of hematopoietic SCT. Bone Marrow Transplant 2011; 46: 1495–1502. lial cells from CsA-induced cell damage (Figure 3). 12 Ikezoe T, Takeuchi A, Chi S, Takaoka M, Anabuki K, Kim T et al. Effect of We have very recently conducted a multi-center clinical trial recombinant human soluble thrombomodulin on clinical outcomes of patients evaluating the effect of rTM on transplantation-associated coagulo- with coagulopathy after hematopoietic stem cell transplantation. Eur J Haematol pathy after HSCT. Intriguingly, prophylactic use of rTM for 2013; 91:442–447. transplantation-associated coagulopathy from day 4 to day 14 after 13 Saito H, Maruyama I, Shimazaki S, Yamamoto Y, Aikawa N, Ohno R et al. Efficacy HSCT significantly decreased the incidence of GVHD as well as SOS and safety of recombinant human soluble thrombomodulin (ART-123) in 30 disseminated intravascular coagulation: results of a phase III, randomized, compared with control patients who did not receive rTM. In double-blind clinical trial. J Thromb Haemost 2007; 5:31–41. addition, a pre-clinical study with a murine GVHD model demon- 14 Tsubokura M, Yamashita T, Inagaki L, Kobayashi T, Kakihana K, Wakabayashi S strated that use of rTM increases the population of regulatory T cells et al. Fatal intracranial hemorrhage following administration of recombinant in the spleen in conjunction with a decrease in the plasma levels of thrombomodulin in a patient after cord blood transplantation. Bone Marrow inflammatory cytokines and significantly prolongs the survival of Transplant 2011; 46:1030–1031. HSCT-received mice.31 One of several other explanations could relate 15 James CM. Pichia protocols 2nd edn. Methods in Molecular Biology Vol. 389. to TME5; rTM could alleviated inflamed immune cells via TME5. The Springer: New York, NY, 2007. fi 16 Yurimoto H, Yamane M, Kikuchi Y, Matsui H, Kato N, Sakai Y. The pro-peptide of rst step of developing GVHD relates to destruction of the intestinal Streptomyces mobaraensis transglutaminase functions in cis and in trans to mucosa, which leads to bacterial translocation from the intestine to mediate efficient secretion of active enzyme from methylotrophic yeasts. Biosci 32 systemic circulation. ThecytoprotectiveactionofTME5couldhave Biotechnol Biochem 2004; 68: 2058–2069. a role in maintaining the integrity of the intestinal mucosa barrier 17 Ikezoe T, Yang Y, Heber D, Taguchi H, Koeffler HP. PC-SPES: a potent inhibitor of function and preventing GVHD. Future experiments will investigate nuclear factor-kappa B rescues mice from lipopolysaccharide-induced the prophylactic effect of TME5 against GVHD in a murine model. septic shock. Mol Pharmacol 2003; 64: 1521–1529. Taken together, the cytoprotective function of TM is localized 18 Ikezoe T, Tanosaki S, Krug U, Liu B, Cohen P, Taguchi H et al. Insulin-like growth factor binding protein-3 antagonizes the effects of retinoids in myeloid in TME5. Use of TME5 may be a promising strategy against leukemia cells. Blood 2004; 104: 237–242. transplantation-associated complications such as SOS and TMA. 19 Niimi S, Oshizawa T, Naotsuka M, Ohba S, Yokozawa A, Murata T et al. Establishment of a standard assay method for human thrombomodulin and determination of the activity of the Japanese reference standard. Biologicals 2002; CONFLICT OF INTEREST 30:69–76. GH is an employee of Asahi Kasei Pharma. The remaining authors declare no 20 Nakamura K, Hatano E, Miyagawa-Hayashino A, Okuno M, Koyama Y, Narita M competing financial interest. et al. Soluble thrombomodulin attenuates sinusoidal obstruction syndrome in rat through suppression of high mobility group box 1. Liver Int 2014; 34: 1473–1487. 21 Shi CS, Shi GY, Chang YS, Han HS, Kuo CH, Liu C et al. Evidence of human ACKNOWLEDGEMENTS thrombomodulin domain as a novel angiogenic factor. 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