An inhibitor of Xa, rivaroxaban was purchased from Tronto Research Chemicals Inc. (Toronto, Canada). Citrated human plasma from Tennessee Blood Sciences, Memphis, TN, USA. Bovine serum albumin (BSA) (essentially fatty acid free and essentially globulin free, lyophilized powder), diphenylene iodonium (DPI) and a blocker of PAR-1, FR171113 were from Sigma (St. Louis, MO, USA). D-glyceraldehyde was purchased from Nakalai Tesque (Kyoto, Japan).
Preparation of AGE-BSA
AGE-BSA was prepared as described previously . In brief, BSA (25 mg/ml) was incubated under sterile conditions with 0.1 M glyceraldehyde in 0.2 M NaPO4 buffer (pH 7.4) for 7 days. Then unincorporated sugars were removed by PD-10 column chromatography and dialysis against phosphate-buffered saline. Control non-glycated BSA was incubated in the same conditions except for the absence of reducing sugars. Preparations were tested for endotoxin using Endospecy ES-20S system (Seikagaku Co., Tokyo, Japan); no endotoxin was detectable.
HUVECs obtained from Lonza Group Ltd. (Basel, Switzerland) were cultured in endothelial basal medium supplemented with 2% fetal bovine serum, 0.4% bovine brain extracts, 10 ng/ml human epidermal growth factor and 1 μg/ml hydrocortisone according to the supplier’s instructions. Rivaroxaban or AGE treatment was carried out in a medium lacking fetal bovine serum, epidermal growth factor and hydrocortisone.
Dihydroethidium (DHE) staining
HUVECs were pre-incubated with or without the indicated concentrations of rivaroxaban for 30 min and treated with 100 μg/ml AGE-BSA, 100 μg/ml non-glycated BSA or 3% citrated human plasma in the presence or absence of 50 nM DPI, 1 mM FR171113 for 4 hr. Then the cells were incubated with phenol red free Dulbecco's Modified Eagle Medium containing 3 μM DHE (Molecular Probes Inc., Eugene, OR, USA). After 15 minutes, the cells were imaged under a laser-scanning confocal microscope. Superoxide generation was evaluated by intensity of DHE staining. The intensity was analyzed by microcomputer-assisted NIH image.
Real-time reverse transcription-polymerase chain reactions (RT-PCR)
HUVECs were pre-incubated with or without the indicated concentrations of rivaroxaban for 30 min and treated with 100 μg/ml AGE-BSA, 100 μg/ml non-glycated BSA or 3% citrated human plasma in the presence or absence of 1 mM FR171113 for 4 hr. Then total RNA was extracted with RNAqueous-4PCR kit (Ambion Inc., Austin, TX, USA) according to the manufacturer’s instructions. Quantitative real-time RT-PCR was performed using Assay-on-Demand and TaqMan 5 fluorogenic nuclease chemistry (Applied Biosystems, Foster city, CA, USA) according to the supplier’s recommendation. IDs of primers for human RAGE, MCP-1, ICAM-1, β-actin and 18S gene were Hs00153957_m1, Hs00234140_m1, Hs00164932_m1, Hs99999903_m1, and Hs99999901_s1, respectively.
Assay of THP-1 cell adhesion to HUVECs
Human THP-1 monocytic leukemia cells (American Type Culture Collection, Manassas, VA, USA) were maintained in RPMI 1640 medium supplemented with 1% GultaMAX (Life Technologies Corporation, Carlsbad, CA, USA) and 1% fetal bovine serum (NICHIREI BIOSCIENCES INC, Tokyo, Japan). THP-1 cells were labeled with 3 mM BCECF-AM (Dojindo, Kumamoto, Japan) at 37°C for 30 min according to the supplier’s recommendation. THP-1 cell adhesion to HUVECs was assayed according to the method described previously . Briefly, HUVECs were treated with or without 100 μg/ml non-glycated BSA or 3% citrated human plasma in the presence or absence of 30 nM rivaroxaban for 4 hr, and then incubated with BCECF-AM-labeled THP-1 cells for 4 hr. After the incubation, nonadherent THP-1 cells were removed by washing the HUVECs gently. Fluorescent intensities of the adherent THP-1 cells were measured.
Western blot analysis
HUVECs were treated with or without 100 μg/ml AGE-BSA or 100 μg/ml non-glycated BSA for 4 hr. Then proteins were extracted from HUVECs with lysis buffer, separated by SDS-PAGE and transferred to nitrocellulose membranes as described previously . Membranes were probed with antibodies raised against PAR-1 (Santa Cruz Biotechnology Inc., Delaware, CA, USA) or α-tubulin (Santa Cruz Biotechnology Inc.), and then immune complexes were visualized with an enhanced chemiluminescence detection system (Amersham Bioscience, Buckinghamshire, United Kingdom).
All values were presented as means ± standard error. Student’s t-test or one-way analysis of variance followed Tukey’s test was performed for statistical comparisons; p < 0.05 was considered significant. All statistical analyses were performed with the use of the PASW Statistics system (version 18.0; IBM Corporation, New York, NY, USA).