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yet, clinical trials evaluating the efficacy of anticoagu- lants or thrombolytics for treatment of endothelial syn- drome have not been conducted.8-10
Since 2008, recombinant human soluble thrombomod- ulin (rTM) has been used to treat disseminated intravas- cular coagulation in Japan. rTM binds thrombin and con- verts protein C to activated protein C (APC), which inhibits activated factor V and VIII and acts as an anti- coagulant.11-14 APC is well known to protect various cell types, including endothelial cells and podocytes, via pro- tease-activated receptor 1 and endothelial protein C receptor.15 rTM counteracted capillary leakage in a patient who developed ES after HSCT.16 In addition, rTM rescues individuals with SOS and TA-TMA developed after HSCT.17-21 We previously showed that rTM possesses the ability to protect vascular ECs in an APC-dependent and ABC-independent manner from various insults, including the calcineurin inhibitor cyclosporine.22,23 In addition, we found that the cytoprotective and pro-angiogenic func- tions of rTM are localized in the fifth epidermal growth factor-like domain of thrombomodulin (TME5), which does not possess the ability to produce APC, although it retains some binding capacity towards thrombin.24 Furthermore, we found that G protein-coupled receptor 15 (GPR15) expressed on vascular ECs is indispensable for the cytoprotective functions of TME5.25,26
TME5 consists of 40 amino acids, including six cysteine residues that form three disulfide bonds, making TME5 a structure with three separate disulfide-bonded loops: the A loop (residues C390 to C395), B loop (residues C399 to C407), and C loop (residues C409 to C421).27 In contrast with the A and B loops, the structure of the C-loop is sim- ilar to that of epidermal growth factor (EGF).27 In the study herein, we identified the minimum structure of TME5 that exerts its cytoprotective and pro-angiogenic activities in vitro and in vivo.
Methods
Cell culture
HUVECs were purchased from Lonza Walkersville Inc. (Walkersville, MD, USA) and cultured in endothelial cell growth basal medium-2 (EBM-2) culture medium supplemented with endothelial cell growth factors (EGM-2; Lonza Walkersville Inc.). Human hepatic sinusoidal endothelial cells (HHSECs) were pur- chased from ScienCell (San Diego, CA, USA) and cultured (37°C, 5% CO2) in endothelial cell medium (ECM, containing 5% fetal bovine serum (FBS; ScienCell).
Murine thoracic aorta vascular ECs were isolated from mice as previously described.28 Briefly, mice were anesthetized, and the thoraces were opened to expose the heart and lungs. The aorta was dissected out and immersed in 20% FBS dulbecco's modi- fied eagle medium (FBS-DMEM; Wako, Tokyo, Japan) in the presence of collagenase type II (Sigma-Aldrich, Tokyo, Japan) for 45 min at 37°C. The cells were then collected and cultured with DMEM supplemented with endothelial cell growth supplement (Sigma-Aldrich, Tokyo, Japan). Five days later, the cells were harvested and utilized for further experiments.
Mice
Gpr15 knockout (Gpr15 KO) mice (129/SvEv; 129P2-Gpr15 tm1.1Litt/J, stock number 008769) were purchased from Jackson Laboratory (Bar Harbor, ME, USA). This strain had been backcrossed to C57BL/6 for three generations before being
TME5C exerts cytoprotective and angiogenic functions
Table 1. Amino acid sequences of TME5A, TME5B, and TME5C.
Name
TME5A
TME5B
TME5C
TME5C mutant
TM: thrombomodulin.
Amino acid sequence
QMFCNQTACPA
DCDPNTQASCE ECPEGYILDDGFICTDIDE ECPEAYILDDGFICTDIDE
haematologica | 2018; 103(10)
used for experiments. Female C57BL/6 mice (8-week-old) were purchased from Japan SLC, Inc. (Hamamatsu, Japan). Female BALB/c (H-2Kd, donor) and female C57BL/6 (H-2Kb, recipient) mice, aged ten weeks and weighing 20-25 g, were purchased from Japan SLC, Inc. All procedures were performed according to the animal care guidelines of Fukushima Medical University. During invasive operations, animals were anesthetized by inhal- ing isoflurane.
Reagents
TM mutants TME5A (residues C387-C397), TME5B (residues C398-C408), and TME5C (residues C408-C426) were synthe- sized by the Peptide Institute Inc. (Osaka, Japan). The TME5C mutant with a single amino acid substitution was synthesized by GL Biochem (Shanghai, China). The amino acid sequences are listed in Table 1. Cyclophosphamide (CY) was purchased from Shionogi & Co., Ltd (Osaka, Japan). Busulfan (BU) and tacrolimus (FK506) were purchased from Sigma-Aldrich, Tokyo, Japan. TME5 and rTM were provided by Asahi Kasei Pharma (Tokyo, Japan).
Proliferation assays
HUVECs (5×103 cells/well), HHSECs (5×103 cells/well), or murine ECs (5×103 cells/well) were cultured in 96-well plates containing TME5C (25, 50, 250, 500, 1000 nM), TME5A (500 nM), TME5B (500 nM), or TME5 (30 nM) with or without FK506 (10 mg/ml) for 24 h. Bromodeoxyuridine (BrdU, 10 mM/well) was added and incubated for an additional 4 h. The quantity of BrdU incorporated into cells was assessed in accordance with the manufacturer’s protocol (Roche, Basel, Switzerland).
Vascular permeability assay
The effects of FK506 and TM mutants on vascular permeabil- ity were measured by a vascular permeability assay kit (Millipore, Billerica, MA). Briefly, HUVECs were plated onto col- lagen-coated inserts and cultured for 72 h until confluence. After starvation for 24 h, cells were treated with TME5A/B/C (500 nM) or TME5 (30 nM) with or without FK506 (10 mg/ml) for 12 h. Then fluorescein isothiocyanate-dextran was added. The extent of permeability was determined by measuring the fluo- rescence of the plate well solution (excitation: 485 nm, emission: 535 nm).
In vitro vascular tube formation assay
To evaluate the pro-angiogenetic effects of TME5C, TME5A,
and TME5B in vitro, HUVECs or HHSECs were plated on growth factor-reduced matrigel (Corning corporation, NY, USA) pre- coated 24-well plates (2.0×104 cells/well) and incubated with control diluent, TME5 (30 nM), TME5A/B/C (500 nM), or vascu- lar endothelial growth factor (VEGF, 0.5 nM, positive control). After 8 h, the endothelial cell-derived tube-like structure was photographed using an inverted microscope (KEYENCE BZ- X700, Osaka, Japan) (magnification ×40). The tube length in three randomly chosen fields from each well was measured using NIH ImageJ software (NIH, Bethesda, MA, USA).
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