Supplementary Material Online
Methods
Study population for human serum specimens
From January 2006 to February 2008, subjects referred to the University of Ottawa Heart
Institute (UOHI) for evaluation of coronary artery disease (CAD) were prospectively enrolled in
a longitudinal cohort study to evaluate the association of serum HSP27 levels with CAD and
prognosis for major adverse cardiovascular events (MACE). Patients enrolled had to be low or
intermediate risk with unknown coronary anatomy and be referred for either invasive coronary
angiography or cardiac computed tomography (CT). Exclusion criteria included diagnosis of
non-ST elevation myocardial infarction, ST elevation myocardial infarction, and cardiac
transplantation.
Significant CAD was defined as the presence of >50% stenosis identified in any major epicardial
vessel. MACE was defined as the occurrence of any myocardial infarction (MI), cerebrovascular
accident (CVA, stroke or transient ischemic attack) or death in follow-up. Myocardial infarction
was defined as a presentation consistent with an acute coronary syndrome and a rise in cardiac
biomarkers >95th percentile and a clinical diagnosis of MI by a treating cardiologist. CVA was
defined as a clinical diagnosis by a treating neurologist. All treating physicians were blinded to
HSP27 levels in adjudicating clinical outcomes. Follow-up included any clinical contacts (by
telephone or clinic visit) at either the UOHI or the Ottawa Hospital. The study complites with the
Declaration of Helsinki, was approved by the UOHI Ethics Institutional Review Board and all
patients provided written informed consent.
HSP27 ELISA
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Serum HSP27 levels were measured using an ELISA kit specific to human HSP27 according to
the manufacturers instructions (QIA119, Calbiochem, San Diego, California) (1, 2).
Experimental animals
All experimental procedures involving laboratory animals were approved by the Animal Care
and Use Committee of the University of Ottawa. We have previously described transgenic mice
that over-express human Heat Shock Protein 27 on an ApoE-/- background (ApoE-/-HSP27o/e) (1,
2). ApoE-/- mice (C57BL/6 background) were purchased from the Jackson Laboratory (Bar
Harbor, Maine). Mice were fed an atherogenic diet consisting of 1.25% cholesterol, 15.8% fat;
(diet TD94059 Harlan Teklad, Madison, Wisconsin) to accelerate atherogenesis. Bone marrow
transplant (BMT) experiments were performed according to previously published protocols (3).
Briefly female ApoE-/- mice (10 week old and ≥ 18.5 g), were housed under sterile conditions
and provided with autoclaved food and water ad libitum a week prior to irradiation. To eliminate
endogenous bone marrow, irradiation was administered through two doses of γ-irradiation, 3-4
hours apart (total of 9 grays using an MDS Nordion Gamma Cell 1000). BMT recipients
received 5×106 bone marrow cells from either female ApoE-/- or ApoE-/-HSP27o/e donors by tail
vein injection. After 6 weeks of recovery recipients were placed on TD94059 ad libitum for 4
weeks to accelerate atherosclerotic lesion development.
For studies of the vascular therapeutic potential of exogenous, recombinant HSP27 (rHSP27) on
lesion progression, subcutaneous (s.c.) injections, twice daily (b.i.d.) of either 100 μg of rHSP27
or PBS were administered to female ApoE-/- mice for three weeks while fed TD94059 ad libitum
. Similarly, we examined the therapeutic potential of exogenous rHSP27 on plaque stabilization
in female ApoE-/- mice who were initially fed TD94059 ad libitum for 4 weeks to promote
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atherogenesis before being switched to a standard rodent chow diet and randomized to treatment
with either 100 μg of rHSP27 or PBS s.c., b.i.d.
Production of recombinant HSP27
Recombinant HSP27 (rHSP27) was produced according to previously described methods (4).
Evaluation of en face atherosclerotic lesion area
The aorta was dissected from the ascending to the thoracic segments, and after removing the
adventitia, pinned and photographed for visualization of lesion burden. Thereafter, the aorta was
opened longitudinally, with the primary incision following the lesser curvature of the arch. To
obtain a flat preparation for imaging, a second incision was made along the greater curvature of
the arch down to the level of the left subclavian artery. Lipid-rich intraluminal lesions were
stained with oil red O and photographed. The en face atherosclerotic aortic lesions were analyzed
by two independent observers blinded to the treatment status of the mice using Image-Pro
software (Media Cybernetics, Silver Spring, Maryland). The extent of atherosclerosis was
expressed as the percentage of surface area of the entire aorta covered by lesions.
Preparation of aortic sinus and evaluation of atherosclerotic lesion area
The top half of the heart containing the aortic root was embedded in paraffin or frozen in TissueTek O.C.T. media. Serial 4-m sections of the aortic sinus including the aortic valve leaflets
were sectioned, beginning at the level where the aortic valve first appears, and stained with
hematoxylin and eosin (H&E), Masson’s trichrome, oil red O, or filipin. Atherosclerotic lesion
areas were analyzed by two observers using Image-Pro software (Media Cybernetics, Silver
Spring, MD). Mean lesion area for each mouse was calculated by averaging measurements from
four cross-sections.
Quantification of macrophage content in atherosclerotic lesions
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Briefly, serial 4-m sections of aortic sinus were blocked with 10% normal horse serum
followed by rat anti-mouse macrophage primary antibody (Mac-2; Accurate Chemical and
Scientific Corp., Westbury, New York, USA) diluted in PBS 1:500 at 4C overnight.
Biotinylated rabbit anti-rat was used as secondary antibody (1:100, Vector Laboratories,
Burlingame, California, USA). Endogenous peroxidase activity was quenched with 3% H2O2.
Antibody reactivity was detected with an ABC kit (Vector Laboratories, Burlingame, California,
USA) and visualized with diaminobenzidine (DAB). Sections were counterstained with
hematoxylin, cleared, and mounted. For indirect immunofluorescence staining, sections were
incubated with a fluorescent rabbit anti-rat IgG antibody (1:100; Vector Laboratories) before
addition of the nuclear fluorochrome Hoechst 33258 (0.1 μg/ml; Sigma). Immunopositive areas
in cross sections were quantified using Image-Pro software according to previously described
techniques (5).
Quantification of smooth muscle cell content in atherosclerotic lesions
Briefly, serial 4-m sections of aortic sinus were blocked with 10% normal horse serum.
Sections were incubated with mouse anti--smooth muscle actin alkaline phosphatase
conjugated antibody (SMA; Sigma, Saint Louis, Missouri, USA) diluted in PBS 1:60 at 4C
overnight. Antibody reactivity was detected by using Fast Red substrate (F4648 Sigma, Saint
Louis, Missouri, USA), resulting in a red-colored precipitate at the antigen site. Sections were
counterstained with hematoxylin and mounted with 30% glycerol/PBS. Immunopositive areas in
cross sections were quantified using Image-Pro software according to previously described
techniques (5).
Quantification of collagen in atherosclerotic lesions
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Picro-sirius red was used to stain for collagen as previously described (6, 7). Collagen bundles
were defined as bright yellow or orange observed under polarizing microscope (Olympus). The
collagen areas and lesion areas in cross sections were quantified using Image-Pro software
according to previously described techniques (5).
TUNEL staining
Apoptotic cells in atherosclerotic lesions were detected using an In Situ Apoptosis Detection Kit
(TUNEL, TACS-XL, TA200, R&D Systems, Inc. Minneapolis, Minnesota) according to the
manufacturer’s instructions.
Quantification of cholesterol crystal clefts in atherosclerotic lesions
H&E and Masson’s trichrome stained serial sections were employed for assessment of
cholesterol crystal deposition in atherosclerotic lesions. Cholesterol clefts were defined as ghostlike needle-shaped spaces that resulted from dissolution of cholesterol crystals during paraffinembedding and tissue processing. In addition, the necrotic area was defined by the presence of
pyknosis, karyorrhexis, or complete absence of nuclei.
Measurement of serum cholesterol levels
Total cholesterol levels were determined using an enzymatic assay kit (Wako Pure Chemical
Industries, Ltd, Osaka, Japan). Pooled serum samples were separated by FPLC to obtain lipid
sub-fractions.
Statistical analyses
All data represent the mean ± SD when normally distributed. Otherwise data is represented as the
median (25th, 75th percentile [interquartile range, or IQR]). Categorical variables are described
as number (%). Statistical analyses were performed using a one-way ANOVA for normally
distributed variables and a Wilcoxon two sample difference test for non-normally distributed
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values. For multiple comparisons of HSP27 levels a Kruskal Wallis non-parametric difference
test was performed. No adjustments were made for multiple comparisons. For analysis of MACE
product limit survival estimates were utilized to calculate unadjusted hazard ratios (HR) which
are reported with 95% confidence intervals (CI). Differences were considered significant at P
values <0.05 (two-tailed). Analyses were performed using SigmaStat for Windows version 3.5
(Systat Software, Inc., IL, USA).
References
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shock protein 27 is mediated by estrogen and competitively inhibits acLDL binding to
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an estrogen-dependent mechanism: role of selective estrogen receptor beta modulation.
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