Page 166 - 2018_10-Haematologica-web

X. Wang et al.
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Figure 1. TME5C stimulates proliferation of endothelial cells. (A). Amino acid sequence alignment of TME5A, TME5B, and TME5C. (B, C). BrdU incorporation assay. HUVECs or HHSECs were cultured with TME5C (25, 50, 250, 500, 1000 nM), TME5A (500 nM), TME5B (500 nM), TME5C mutant (500 nM), or TME5 (30 nM) for 24 h. Proliferation was measured by BrdU incorporation assays. Experiments were performed three times in triplicate plates. Results represent the mean ± SD. *P<0.05. BrdU: bromodeoxyuridine; HUVECs: human umbilical vein endothelial cells; HHSECs: human hepatic sinusoidal endothelial cells; TM: thrombomodulin; N.S.: not significant.
Murine angiogenesis assay
To assess the pro-angiogenetic effects of TM mutants in vivo, growth factor-reduced matrigel (0.3 mL, containing 40 U/mL heparin) with control diluent, TME5A/B/C (500 nM), TME5 (30 nM), VEGF (0.5 nM, positive control) was subcutaneously inject- ed into C57BL/6 mice (8-week-old, female) near the abdominal midline. Four days later, mice were euthanized, and the matrigel plugs were dissected out and photographed.
Hemoglobin determination of matrigel plugs
Matrigel plugs were mixed with 1 ml distilled water and put on ice for 5-10 min. After centrifugation for 6 min at 8000 g, the supernatants were mixed with drabkin’s reagent (Sigma-Aldrich, Tokyo, Japan) and hemoglobin was measured as previously described.24 Absorbance was measured with a microplate reader at 540 nm. Methemoglobin (Sigma-Aldrich, Tokyo, Japan) was used to obtain a standard curve.
Apoptosis Assays
The ability of TM mutants to rescue HUVECs, HHSECs, or murine ECs from FK506-induced apoptosis was measured using the Annexin V Apoptosis Detection Kit (K129, BioVision, Milpitas, CA, USA) and propidium iodide (PI) as previously described.22 Briefly, cells were exposed to FK506 (10 mg/ml) with or without TME5A/B/C (500 nM) or TME5 (30 nM). After 36 h, cells were harvested and subjected to PI and PE-Cy5 anti-annex- in V. Early apoptosis cells are annexin V positive and PI negative. Late apoptosis cells are annexin V positive and PI positive.
Western blot analysis
Western blot analysis was performed as described previous- ly.22 The following antibodies were used: anti-p-ERK (T202/Y204) (Cell Signaling Technology, Danvers, MA, USA),
anti-ERK (Cell Signaling Technology; 9102), anti-p-AKT (Ser473) (Cell Signaling Technology; 9271), anti-AKT (Cell Signaling Technology; 9272), anti-p-Stat5 (Tyr694) (Cell Signaling Technology; 9351), anti-Stat5 (Cell Signaling Technology; 9363), anti-p38 (Cell Signaling Technology; 9212 ), anti-p-p38 (Tyr180/182) (Cell Signaling Technology; 9216), anti-Mcl-1 (Cell Signaling Technology; 4572), and anti-GAPDH (Cell Signaling Technology; 5174).
Prothrombin time (PT) and activated partial thromboplastin time (APTT)
For PT detection, 200 ml PT reagent (Sysmex Corporation, Kobe, Japan) was mixed with 100 ml human plasma with or without rTM, TME5, TME5A/B/C, or TME5C mutant (10 ml). For APTT detection, 100 ml APTT reagent (Sysmex Corporation) was mixed with 100 ml human plasma with or without rTM, TME5, TME5A/B/C, or TME5C mutant (10 ml). After incubation for 120 s, 100 ml CaCl2 was added to this mixture. The clotting time was measured using a KC1 Delta coagulometer (Tcoag, Co. Wicklow, Ireland).
SOS murine model
C57BL/6 mice were randomly divided into three groups (n=16 in each group): mice that received vehicle phosphate buffered saline (PBS) without bone marrow transplantation (BMT) were defined as the control group. Mice that received BU/CY followed by BMT and treated with vehicle PBS were defined as the BMT group. Mice that received BU/CY followed by BMT and treated with TME5C were defined as the BMT treated with TME5C group. SOS was induced by previously reported BU/CY reconditioning treatment followed by BMT with some modification;29 in brief, BU (25 mg/kg/day for 4 days) followed by CY (100 mg/kg/day for 2 days) were given to
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haematologica | 2018; 103(10)