Supplemental Digital Content 2: Materials and Methods
The specific PAI-1 inhibitor
A PAI-1 inhibitor (IMD-1622; 3-(3,4-dichloro-benzyl)-5-(3,4,5-trihydroxy
-benzylidene)-thiazolidine-2,4-dione) was designed using a virtual screening
technology of protein structure and made by the Institute of Medical Molecular
Design, Tokyo, Japan.
To confirm the specificity of IMD-1622, an inhibitory assay
against serin proteinase inhibitor (SERPIN) proteins (PAI-1, PAI-2, AT-3,
alpha1-antitrypsin, alpha1-antichymotrypsin and HC2) was performed. Briefly, 1
micro L of DMSO or 100X compound was dispensed into 24 micro L of assay buffer
in a 96-well black plate and agitated for 10 min. 25 micro L of 4X SERPIN and 25
micro L of 4X protease in assay buffer was added and mixed. After 10 min
incubation, protease reaction was started by adding 25 micro L of fluorogenic
substance to the assay buffer. Fluorescence was kinetically monitored at 360 nm
excitation and 465 nm emission in 5-minute intervals for 30 min at an ambient
temperature. Percent inhibition was calculated as follows: % SERPIN inhibition =
100 x (fluorescence with test compound and SERPIN - fluorescence with DMSO and
SERPIN)/(fluorescence without SERPIN - fluorescence with DMSO and SERPIN).
The drug was dissolved in 0.5% carboxymethylcellulose (CMC) vehicle (Sigma,
Tokyo, Japan) immediately before use. Drug-free vehicle (0.5% CMC solution) was
used as a control (7, 8).
Murine Cardiac Transplantation
The male BALB/c and C57BL/6 mice (4-6 weeks, 20-25 g) combination was used as
the major mismatch group for the study of graft survival; the male Bm12 and
C57BL/6 mice (4 to 6 weeks, 20-25 g) combination was used as the class II mismatch
group for pathological analysis of GAD (29-30). They were obtained from Japan
Charles River Laboratories (Tokyo, Japan) and were anesthetized by intraperitoneal
injection of 3.6 % chloral hydrate (0.2 ml / 20 g). Ischemic time averaged 60 min,
and the overall success rate was greater than 95%. The transplanted mice in total
allomismatch combination were assigned randomly to two groups; mice were
treated daily with 20 mg/kg/day of the PAI-1 inhibitor intraperitoneally (n=6); the
other mice (n=6) received the same volume of vehicle for the control. Graft
survival was assessed by graft palpation. The mice with class II mismatch
combination were also assigned randomly to two groups; mice were treated daily
with 20 mg/kg/day of the PAI-1 inhibitor intraperitoneally (n=10); the other mice
(n=10) received the same volume of vehicle for the control. The class II mismatch
allografts were harvested at day 60 after transplantation. During the observation
period, we investigated whether adverse effects (bleeding complications, insufficient
response to infections, etc.) occurred or not. This investigation conforms with the
Guide for the Care and Use of Laboratory Animals in the Tokyo Medical and Dental
University and University of Tokyo.
Histological Examination and Morphometry
Isografts and allografts of the class II mismatch group were harvested on day 60
after transplantation for analysis of GAD and myocardial cell infiltration. Grafts
were sectioned transversely at the maximal circumference of the ventricle. As
previously described, serial sections (6 µm) from tissue embedded in OCT were
stained with hematoxylin eosin (HE) and Elastica van Gieson (EvG) to highlight the
internal elastic lamina (IEL). The grafts were photographed, videodigitized and
processed using an image analysis system (NIH Image). The area encompassed by
the lumen and IEL was traced carefully, and the area of luminal stenosis in each
cross section was calculated according to the formula: luminal occlusion = (IEL area
- luminal area)/IEL area. Scores of two independent reviewers were averaged (3, 5,
30).
Immunohistochemistry
Serial sections (6 µm) were cut and dipped in cold acetone for 10 minutes. The
sections were rehydrated in PBS and incubated with 5% normal goat serum to block
non-specific reactions.
Sections were incubated with primary antibody against
murine CD4, CD8, CD11b (BD Biosciences Pharmingen, San Diego, CA),
intercellular adhesion molecule (ICAM)-1 (YN1/1.7), proliferating cell nuclear
antigen (PCNA) (Santa Cruz Biotechnology Inc., Santa Cruz, CA) or fibrinogen
(A0080, Dako) for 12 h at 4 C.
Antibody-biotin conjugate was detected with
Vectastain ABC Kit (Vector, Burlingame, CA) used according to the manufacturer's
instructions. Enzyme activity was detected with diaminobenzidine (0.5 mg/ml)
with 0.05% NiCl2 in 50 mM Tris buffer, pH 7.5. Immunohistochemical analyses
were performed by independent observers using qualitative scoring as previously
reported (0, absent; 1, weak, focal; 2, weak, diffuse; 3, strong, focal; 4, strong, diffuse,
and 5 very strong and diffuse). Scores uniformly fell within one grade of each
other and were averaged (29, 30).
Zymography
Tissues were snap-frozen in liquid nitrogen and homogenized in 0.5ml of ice-cold
lysis buffer (containing 50mM Tris-HCl, pH6.8, 150mM NaCl, 1mM PMSF, 2ng/ml
aprotinin, 1% Triton X-100, 1% SDS, 1% sodium deoxycholate). Samples were
centrifuged for 10 minutes at 4°C at 20,000G and the supernatant was transferred to
clean new tubes. Protein concentration was determined by use of a BCA protein
assay (Pierce Biotechnology). The extracted samples were stored at -80°C until use.
Protein samples (each 25 micro g) were adjusted to 15 microL, and mixed with an
equal volume of 2×loading buffer (containing 125mM Tris-HCl, pH6.8, 20% glycerol,
4% SDS, 0.05% bromophenol blue), and incubated for 10 minutes at room
temperature. 6% gelatin gel was run at 80V until the bromophenol blue tracking
dye reached the bottom of the gel. The gel was incubated in zymogram renaturing
buffer (Invitrogen Corp, Carlsbad, CA) for 30 minutes, and replaced with fresh
zymogram developing buffer (Invitrogen) and incubated at 37 C for 16 hours. The
gel was stained with 0.2% Coomassie blue G 250 for 2 hours and destained with
methanol: acetic acid: water (5: 1: 4) for 30 minutes. The quantitative scoring was
performed using the Image J software (NIH).
Real-time RT-PCR
Total RNA was isolated from the hearts or cultured cells and cDNA was prepared
with the RT-PCR Kit (Stratagene Co., La Jolla, CA). Real-time PCR in a StepOne
real-time PCR system (Applied Biosystems) was used to determine the mRNA
expression of interferon (IFN)-gamma (Assay ID: Mm00801778_m1), tumor necrosis
factor (TNF)-alpha (Assay ID: Mm00443258_m1), interleukin (IL)-2 (Assay ID:
Mm00434256_m1), IL-6 (Assay ID: Mm00446190_m1), IL-10 (Assay ID:
Mm00439616_m1), monocyte chemoattractant protein (MCP)-1 (Assay ID:
Mm00441242_m1), IFN-gamma-inducible protein of 10 kD (IP-10) (Assay ID:
Mm00445235_m1), macrophage inflammatory protein (MIP-1) alpha (Assay ID:
Mm00441258_m1), MIP-1 beta (Assay ID: Mm00443111_m1), PAI-1
(Mm00435858_m1) and 18s rRNA (Assay ID: Hs99999901_s1) as a control.
The
real-time PCR protocol consisted of an initial step at 95°C for 20 sec followed by 50
cycles: 95°C for 1 sec, annealing at 60 °C for 20 sec. cDNA was run in duplicates
and quantitative data was calculated using the comparative CT (CT) method.
Cell Proliferation Assay
Single-cell splenocyte suspensions were prepared by dissociating tissue with frosted
glass slides. A total of 5 X 105 naive responder cells were cultured with an equal
number of mitomycin-C treated stimulator cells in 96 well plates in C/10 media.
The PAI-1 inhibitor was added to each well at various concentrations (low, 10-10 M;
middle, 10-9 M; and high, 10-8 M). Proliferation was assessed at 37C under 5% CO2
on days 2 through 4. Cell proliferation was assessed with the Cell Counting Kit-8
(Dojindo, Tokyo, Japan). Cell proliferation was expressed as the optical density
(29).
Enzyme-linked Immunosorbent Assay (ELISA)
Supernatant was collected from cultures used for cell proliferation assay.
Concentrations of IFN-gamma were determined with ELISA kits (BioSource
International, Camarillo, CA) according to the manufacturer’s instructions (29).
Statistical Analysis
All data are expressed as mean +/- SD.
Scores were compared among the groups
using a Scheffe's ANOVA. Differences with values of P < 0.05 were considered
significant.