Recombinant Human Thrombomodulin Suppresses Experimental Abdominal Aortic
Aneurysms Induced by Calcium Chloride in Mice
(Supplementary Data)
Chao-Han Lai, MD1,2,4, Guey-Yueh Shi, PhD2,3, Fang-Tzu Lee, MS2,3, Cheng-Hsiang Kuo,
PhD2,3, Tsung-Lin Cheng, PhD2,3, Bi-Ing Chang, MS2,3, Chih-Yuan Ma, PhD2,3, Fu-Chih Hsu,
MS2,3, Yu-Jen Yang, MD, PhD2,4, Hua-Lin Wu, PhD2,3
From the 1Institute of Clinical Medicine, 2Cardiovascular Research Center, 3Department of
Biochemistry and Molecular Biology, and 4Department of Surgery, National Cheng Kung
University Hospital, College of Medicine, National Cheng Kung University, Tainan, Taiwan
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SUPPLEMENTARY METHODS
Assays for rTMD123-HMGB1 interaction
For co-immunoprecipitation assay, aortic homogenates obtained 3 days after AAA
induction were co-incubated with c-myc tagged rTMD123 (50 g), rabbit anti-HMGB1
polyclonal antibody (Abcam, Cambridge, MA), and protein A agarose beads (Millipore,
Billerica, MA) overnight at 4C in immunoprecipitation buffer (50 mM Tris-HCl, 150 mM
NaCl, 1% NP-40, and 1 mM PMSF; final volume: 500 l). Rabbit IgG antibody was served
as a negative control. Beads were washed with phosphate-buffered saline (PBS) containing
0.05% Tween-20 three times and the immune precipitates were analyzed using western blot
analysis followed by immunoblotting with mouse anti-c-myc monoclonal antibody (Santa
Cruz Biotechnology, Santa Cruz, CA). For analyzing the binding ability of HMGB1 to
THP-1 cells, THP-1 cells were co-incubated with human recombinant HMGB1 protein (R&D
systems, Minneapolis, MN) and rTMD123 for 30 min at 4C. Cells were washed with
ice-cold PBS three times, stained with Alex Fluor 488-conjugated goat anti-rabbit polyclonal
antibody (Invitrogen, Carlsbad, CA) for 30 min at 4 C, and then were stained with rabbit
anti-HMGB1 polyclonal antibody for 30 min at 4C. Cells were analyzed by
fluorescence-activated cell sorting (FACS; BD Biosciences, San Jose, CA). The geometric
mean fluorescence intensity was determined by WinMDI 2.9 software.
Quantification for proinflammatory mediators and MMPs in the aortic wall
The levels of HMGB1 and RAGE in the aortic wall were analyzed using western blot
analysis. Equal protein amounts of mouse aortic homogenates were resolved on 10%
SDS-PAGE gels and were transferred to nitrocellulose by electroblotting. The membrane was
blocked with Tris-buffered saline and Tween containing 5% nonfat dry milk to prevent
nonspecific antibody binding. After being probed with appropriate dilution of antibodies
against RAGE (Santa Cruz Biotechnology) and HMGB1 (Abcam) overnight at 4°C, the
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membrane was incubated with a horseradish peroxidase-conjugated secondary antibody for 1
hour at room temperature. The signal was detected using an enhanced chemiluminescence
reagent (Amersham Phamacia Biotech, Piscataway, NJ). The band intensity was
quantitatively determined using Gel-Pro Analyzer software. Blots were reprobed with β-actin
(Sigma-Aldrich, St Louis, MO) to confirm equal protein loading.
Mouse aortic homogenates were also analyzed using commercially available ELISA kits
specific for mouse TNF-α (R&D systems), IL-6 (R&D systems), MCP-1 (R&D systems),
total MMP-9 (Abnova, Heidelberg, Germany) and total MMP-2 (Abnova) according to the
manufacturers’ protocols. Each of these assays uses a dual-antibody method with a reported
sensitivity of 3.9 pg/mL. The amount of each protein in each sample was measured by
spectrophotometric optical density (450 nm) using an automated microplate reader
(SpectraMax 340PC384; Molecular Devices, Sunnyvale, CA).
SUPPLEMENTARY FIGURE LEGENDS
SUPPLEMENTARY FIGURE 1. A, rTMD123 can interact with HMGB1 in the aorta.
Fifty-μg rTMD123 protein (tagged with c-myc), rabbit anti-HMGB1 antibody, and protein A
beads were added in the aortic homogenates (obtained on day 3) overnight at 4C. Rabbit IgG
antibody (IgG) was served as a negative control. Beads were washed thrice with PBS
containing 0.05% Tween-20 and the immune precipitates were analyzed by western blot. The
result is typical of those obtained in 3 independent experiments. B, rTMD123 inhibits
HMGB1 binding to THP-1 cells in a concentration-dependent manner. THP-1 cells were
co-incubated with recombinant HMGB-1 protein and rTMD123 for 30 min at 4C. Cells
were washed, stained with rabbit-anti HMGB-1 antibody for 30 min at 4C, and were stained
with Alexa Fluor 488-conjugated goat anti-rabbit antibody for 30 min at 4C. Cells were
analyzed by FACS. The geometric mean fluorescence intensity was determined by WinMDI
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2.9 software (n=3). (**P<0.01 compared with untreated group. #P<0.05 compared with
HMGB1-treated group.)
SUPPLEMENTARY FIGURE 2. rTMD123 attenuates HMGB1-induced MMP-9 in
differentiated THP-1 cells. A, activities of MMP-9 and MMP-2 were assessed after HMGB1
treatment for 24 hours (n=4). B, MMP-9 activity was assessed after sRAGE pretreatment for
1 hour and HMGB1 treatment for 24 hours (n=4). C, MMP-9 activity was assessed after
rTMD123 pretreatment for 1 hour and HMGB1 treatment for 24 hours (n=4). (All activities
were assessed by gelatin zymography. *P<0.05, **P<0.01, n.s. P>0.05 compared with
untreated group. #P<0.05 compared with HMGB1-treated group.)
SUPPLEMENTARY FIGURE 3. Histological analysis for aortic samples obtained on day 7
showed that rTMD123 treatment attenuates macrophage infiltration in the aortic wall (A) and
apoptosis in the media (B) preceding AAA formation. (P=AAA-PBS, rTM=AAA-rTM.
*P<0.05, ***P<0.001 compared with sham. #P<0.05, ###P<0.01 compared with AAA-PBS.
L indicates lumen. All scale bars represent 100 μm. White arrows indicate examples of
MOMA-1-positive, DAPI-stained macrophages. Red arrows indicate TUNEL-positive cells.)
SUPPLEMENTARY FIGURE 4. Aortic samples obtained on day 28 showed that rTMD123
treatment reduces the activities of MMP-9 and MMP-2 in the aortic wall measured by gelatin
zymography (n=4). (P=AAA-PBS, rTM=AAA-rTM. **P<0.01 compared with sham.
#P<0.05 compared with AAA-PBS.)
SUPPLEMENTARY FIGURE 5. Treatment with rTMD123 preserves elastin integrity and
SMCs in the aortic wall 28 days after AAA induction. A, medial elastin degradation (n=6).
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Elastin degradation grading scales (4 grades) by VVG staining are indicated in right panels
(Blue arrows indicating disrupted elastic lamella). B, SMC content (n=6). SMC content
grading scales (4 grades) by α-SMA staining are indicated in right panels. (P=AAA-PBS,
rTM=AAA-rTM. **P<0.01, ***P<0.001 compared with sham. #P<0.05 compared with
AAA-PBS. L indicates lumen. All scale bars represent 50 μm.)
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