Secreted Frizzled Related Protein 4 Reduces Fibrosis
Scar Size and Ameliorates Cardiac Function
After Ischemic Injury
Kentaro Matsushima, B.A.,1,2Takashi Suyama, B.A.,1,2Chiemi Takenaka, B.A.,1,3Naoki Nishishita, Ph.D.,1,3
Keiko Ikeda, Ph.D.,4Yoshito Ikada, Ph.D.,5Yoshiki Sawa, M.D., Ph.D.,6Lars Martin Jakt, Ph.D.,3
Hajime Mori, Ph.D.,4,7and Shin Kawamata, M.D., Ph.D.1,3
We show that intramuscular administration of recombinant Sfrp4 to rat heart ischemic injury and recanalization
least 20 weeks when Sfrp4 was administered in a slow release system (Sfrp4-polyhedra) to both acute and subacute
ischemic models. The histology of the dissected heart showed that the cardiac wall was thicker and the area of
acellular scarring was smaller in Sfrp4-treated hearts than in controls. Increased amounts of both the inactive serine
9-phosphorylated form of glycogen syntase kinase (GSK)-3b and the active form of b-catenin were observed by
immunohistology 3 days after lateral anterior descendant ligation in control, but not in Sfrp4-treated hearts. All
together, we show that administration of Sfrp4 interferes with canonical Wnt signaling that could mediate the
formation of acellular scar and consequently contributes to the prevention of aggravation of cardiac function.
caused by myocardial infarction can result from either acute
loss of heart function immediately after the infarct or chronic
heart failure after initial survival (due to either the mildness
of the infarct or successful medical intervention). Heart
pump function lost after ischemic injury cannot be recovered
by conventional medication, and the ischemic heart has
therefore been thought to be an ideal target for regenerative
medicine utilizing stem cell transplantation. However, trans-
plantation of putative tissue-derived cardiac progenitors have
failed to produce any promising clinical benefits.1–4Cardiac
progenitors derived from ES cells have been shown to provide
a small improvement in cardiac function in animal ischemic
models5and such therapies may become useful in the future.
Presently, however, several critical issues (e.g., the cell puri-
fication process, low cell survival, and immune rejection) re-
main to be resolved before such cells can be used in the clinic.6
These facts prompted us to search for secreted factors that
he consequences of myocardial infarction remain the
leading causes of death in the developed world. Death
underlie cardiac functional recovery and that might im-
prove cardiac function after ischemic injury using micro-
We have previously made use of gene expression data
from both a mouse myocardial infarction model7and a rat
infarction and treatment model8to identify genes (a) that are
upregulated after infarction, (b) whose expression is further
increased as a result of myoblast sheet application (which
provides therapeutic benefit),9and (c) that encode secreted
factors that have the potential to act when applied exoge-
nously to the heart. Secreted frizzled related protein 4 (Sfrp4),
Midkine, and pleiotrophin all met these criteria (Supplemental
Table S1, available online at www.liebertonline.com/ten). We
have previously reported that application of Midkine to is-
chemic areas can partially block postinfarct deterioration of
heart function9; however, of the remaining genes, only Sfrp4
showed obvious effects in a preliminary screen; hence in this
work we focused on the therapeutic potential of Sfrp4.
Sfrp proteins are characterized by a frizzled-like cysteine-
rich domain (CRD) in the N-terminal half and form a family
of soluble proteins (Sfrp1-5) that can be subdivided into two
1Foundation for Biomedical Research and Innovation TR1308, Kobe, Japan.
2Bay Bioscience Corp., Kobe, Japan.
3Center for Developmental Biology, Kobe, Japan.
4Protein Crystal Corp., Osaka, Japan.
5Department of Indoor Environmental Medicine, Nara Medical University, Kashihara city, Nara, Japan.
6Division of Cardio-Vascular Surgery, Department of Surgery, Osaka University Graduate School of Medicine, Osaka, Japan.
7Department of Applied Biology, Kyoto Institute of Technology, Kyoto, Japan.
TISSUE ENGINEERING: Part A
Volume 16, Number 11, 2010
ª Mary Ann Liebert, Inc.
groups; Sfrp1, Sfrp2, Sfrp5, and Sfrp3, and Sfrp4 on the basis
of sequence homology.10Frizzled proteins act as receptors
for Wnt ligands and it is thought that the Sfrps may also
interact with Wnt proteins.11The expression patterns of the
Sfrps during chick, mouse, and xenopus embryonic devel-
opment overlap with their Wnt counterparts,12–14supporting
the idea that Sfrps modulate Wnt signaling through the
frizzled-like CRDs.15,16However, it remains unclear whether
Sfrps physiologically interact with Wnts through the CRD or
even whether they act as Wnt antagonists or agonists.17,18
Additionally, since many Wnt proteins can interact with
several different Frizzled receptors, it is still unclear which
ligand–receptor interactions are physiologically relevant and
what signaling results from which interactions.
Heart ischemic injuries can result from either permanent
or temporary occlusion of cardiac arteries. Although these
injuries have different pathologies, both result in the death of
cardiomyocytes and the replacement of cardiac tissue with
scar tissue. Permanent ischemic injuries result in cell death
due to the resulting anoxic conditions, whereas transient is-
chemia is believed to cause secondary tissue damage
through the diffusion of inflammatory factors from the is-
chemic area (reperfusion injury). Patients who have under-
gone per-cutaneous trans-coronal angioplasty intervention
after coronary occlusion frequently present physiological
conditions characteristic of reperfusion injury. Permanent
ischemic damage can be modeled using a ligation of the
lateral anterior descendant (LAD) branch, whereas transient
ischemia can be modeled by a temporary LAD ligation ap-
plied during the period of surgery.19
Both transient and permanent ischemic injuries result in
cell death, scar formation, and a tissue remodeling process
that continues over a period longer than 1 month.20It
therefore seems likely that a single application of a protein
product to the ischemic area will only have an effect during
the early stages of tissue remodeling, and make it desirable
to identify means of providing a long-term delivery of pro-
tein products to specific areas of tissues. It has previously
been shown that proteins can be immobilized within insect
viral particles (polyhedrons). Such particles are slowly de-
graded by extracellular proteases, resulting in the gradual
release of the immobilized proteins.21,22Polyhedra particles
may thus facilitate the in vivo long-term delivery of thera-
peutic proteins to injured tissues.
In this report, we demonstrate for the first time that the
Wnt signal modulator Sfrp4 is upregulated in ischemic heart
and that administration of Sfrp4 improves cardiac function
after both permanent and transient ischemic injury. We fur-
ther show that the therapeutic effect can be prolonged by the
immobilization of Sfrp4 in polyhedra particles and demon-
strate a number of different means by which such particles
can be applied to ischemic regions. Our data suggest that the
therapeutic effect of Sfrp4 application is due to a reduction in
acellular scar tissue formation resulting from an inhibition of
canonical Wnt signaling.
Materials and Methods
Rat heart ischemic and recanalization models
All animal experimental protocols were reviewed by
the animal experiment committee of the Foundation for
Biomedical Research and Innovation. Seven-week-old male
Sprague-Dawley rats (220–250g; Japan SLC) were used to
generate a heart infarction model by ligating the left coro-
nary anterior descendent branch (LAD ligation model) or a
recanalization model by ligating LAD for 1h followed by
release of the ligation. Both ischemic and recanalization
models were generated by open chest surgery as previously
reported.7For the anesthesia of the rats, 2.5mL of anesthetic
solution (10% v/w Ketamine [Ketalar, Daiichi-Sankyo Pro-
pharma]) and 2.5% v/w Xylazine (Selactar, Japan Bayer
Medical) in an isotonic solution in Solita T1 (Shimizu Phar-
maceutical Co.) was administered to the rats intraperitone-
ally. A respiratory device (Respirator SN-480-7x2T; Shinano)
was used to aid the ventilation during the open chest LAD
Sfrp4 was administered to ischemic regions by three dif-
ferent means: (1) by intramuscular (IM) injection of soluble
protein, (2) by IM injection of Sfrp4 containing polyhedra
(S-PH), and (3) by combining S-PH with a biodegradable
Soluble IM injection.
Sfrp4 (sFRP-4; R&D Systems, Inc.) in 50mL phosphate-buff-
ered saline (PBS) was mixed with 50mL collagen type I gel
(BD Biosciences) and injected to the ischemic border zones.
Satisfactory injection of reagents with 26G needles (Terumo)
was confirmed several times by injecting blue ink and ob-
serving ink staining in wall muscle after sacrifice.
Five or 20mg recombinant human
man Sfrp4 gene (CR 541755) was amplified by polymerase
chain reaction (PCR) with the 50Sfrp4 and 30Sfrp4 primers.
Human Sfrp4 protein was then translated as a fusion pro-
tein with VP3, which facilitates immobilization within
polyhedra, to generate S-PH in S9 cells as described.8,9We
estimate that the average mass of Sfrp4-VP3 immobilized in
a polyhedron (one cube) is *8?10?3ng (the average volume
of a polyhedron is 5?5?5mm3with a density of 1.3g/mL
and *5% of the total mass corresponds to Sfrp4-VP3).21,22
Hence 2.5?106polyhedra is roughly equivalent to 20mg of
recombinant protein. Polyhedra cubes were applied in a
number of ways: (1) 2.5?106cubes of polyhedra were mixed
with 100mL of PBS and injected to ischemic border zones, (2)
5?106cubes of polyhedra were immobilized on a 9?9mm
collagen type I sheet by air-drying and applied to the
ischemic heart just after LAD ligation, or (3) 5?106cubes of
S-PH were mixed with 150mL of fibrin glue Bolheal (Teijinn
Pharma) and plastered to the ischemic area directly by
reopening the chest 2 weeks after LAD ligation.
The open reading frame of the hu-
Echocardiographic assessment of cardiac function
Rat left ventricular (LV) functions were echocardio-
graphically monitored with a SONOS 7500 (Philips Electro-
nics). Two operators performed the ultrasound measurement
in a blind fashion. The LV end-systolic area, LV end-diastolic
area (LVEDA), LV dimensions at end-systole, and LV di-
mensions at end-diastole (LVDd) were determined with the
device. Ejection fraction (EF) was calculated by the modified
Simpson method (disc method). The mean of measured long
3330 MATSUSHIMA ET AL.
axis (L) in several cycles was used for the calculation of EF.
LV percent fractional shortening (% FS) was calculated by
dividing the difference between the LVDd and end-systolic
dimension with LVDd, and shown as percentage.
Functional area change (%) was calculated by dividing the
difference between LVEDA and LV end-systolic area with
LVEDA, and shown as percentage. The statistical signifi-
cance of treatment effects was evaluated using repeated
measures analysis of variance (ANOVA) as described below.
Release of Sfrp4 in vitro and in vivo
Serum was obtained from normal (no ischemic injury),
control (ischemic injury followed by application of PBS or
empty polyhedra particles), and Sfrp4-treated rats 1 or 10
weeks after control or Sfrp4 application (for fibrin glue
samples there was a 2 week lag between ischemic injury and
treatment). About 100mL of rat serum was immobilized on
the bottom of the ELISA plate, followed by detection with
anti-Sfrp4 antibody (R&D System AF1827) and anti IgG-HRP
(2nd antibody; R&D System, HAF019). The release of Sfrp4
from S-PH was estimated by fixing 5?103or 5?104cubes of
S-PH to the bottom of 24-well plate wells followed by the
determination of Sfrp4 concentration by ELISA after culture
of 1.5?105primary rat cardiomyocytes in the precoated wells
with 500mL of Dulbecco’s modified Eagle’s medium sup-
plemented with 20% fetal calf serum.
Quantitative reverse transcriptase
polymerase chain reaction
Total RNA from the LV myocardial tissue blocks was
extracted with the RNeasy mini kit (QIAGEN). Quantitative
reverse transcriptase (qRT)-PCR was performed with an ABI
PRISM 7000 (Life Technologies) using SYBR Premix EX
Taq? (RR041A; Takara) in accordance with the manufac-
turer’s instructions. Primers used are listed in Supplemental
Table S2 (available online at www.liebertonline.com/ten).
Expression measurements were normalized with respect to
glyceraldehyde 3-phosphate dehydrogenase expression.
Myocardial tissues were stained with a number of primary
antibodies (Supplemental Table S2). Antibodies were ob-
servedwith the ABC detection kit (Vector Laboratories).
Cell proliferation was measured by in vivo BrdU incor-
poration. Ten mg of BrdU (Sigma) was administered to rats
via intraperitoneal injection 3 days after LAD ligation and
heart sections were prepared the next day (24h labeling).
BrdU incorporation was detected with the BrdU In situ De-
tection kit (BD Pharmingen; #550803). The number of
BrdU(þ) cells was determined in three randomly chosen
areas (300?400mm) lying in the ischemic border areas of
three PBS- or Sfrp4-treated rats (total 9 areas).
The vascular density in infarcted border areas 3 days after
LAD ligation was evaluated by scoring the number of von
Willebrand Factor (vWF)-expressing vessel-like structures
in nine randomly selected areas (300?400mm) from three
PBS- or Sfrp4-treated rats (three areas/rat). Frozen sec-
tions were stained with anti-human vWF antibody (SIGMA:
A-254, 1:100 dilution) followed by detection with donkey
anti-rabbit IgG peroxidase-linked-specific F(ab’)2 fragment
(GE Healthcare:NA9340, 1:100 dilution). The peroxidase
substrate kit DAB (VECTOR:SK-4100) was used for the de-
tection of vWF. Nuclei were stained with Mayer’s hemalum
solution (E.MERCK:1.09249.0500) for microscopic observation.
Measurement of left ventricle wall thickness
and percent fibrosis
Horizontally sectioned rat heart slices were stained by
Masson’s Trichrome. The wall thickness of sectioned hearts
in ischemic area was measured in a blind fashion at 4 dif-
ferent points in a total of 10 rats (4 points/rat) for each
treatment group. The percentage of fibrous area in ischemic
regions was determined by calculating the area of positive
Masson’s Trichrome staining region (blue) as a proportion of
the total ischemic area utilizing an Adobe Photoshop
(www.adobe.com) two-value recognition function. The pro-
portion of fibrous area was measured in four rats for each
All statistical analyses were performed using the indicated
functions and packages in the R software environment
All EF data were normalized by preinjury values (i.e.,
expressed as a fraction of the preinjury EF for that rat) to
minimize noise caused by differences between individual
rats. Postinjury EFs and standard errors are shown in Sup-
plemental Fig. S1 (available online at www.liebertonline
.com/ten). Individually normalized EF data were then ana-
lyzed using repeated measures ANOVA as implemented in
the ‘‘ANOVA’’ function of the ‘‘car’’ R package. We used the
‘‘ANOVA’’ function to test for the presence of treatment and
treatment–time interaction effects. The ‘‘ANOVA’’ function
performs both type II repeated measures multivariate AN-
OVA and univariate type II repeated-measures ANOVA
with corrections for departures from sphericity. Since the
inference of F-values from multivariate ANOVA is nontriv-
ial, the ‘‘ANOVA’’ function reports 4 different p-value esti-
mates based on different means of estimating the degrees of
freedom within the data set. Hence, the ‘‘ANOVA’’ function
reported 4 multivariate ANOVA p-value estimates (Pillai,
Wilks, Hotelling-Lawley and Roy for treatment and treat-
ment-time interaction) and 4 univariate p-value estimates
(raw treatment and treatment-time interaction as well as two
different corrections) (Greenhouse-Geisser and Huynh-Feldt)
for departures from sphericity. In the cases where more than
two treatment groups were present in the data set, we used
models containing specific subsets (depending on the pur-
pose of the experiment) of the data to determine significant
differences between treatments. Although this could lead to
type I errors, the p-values were sufficiently low to withstand
Bonferroni corrections for multiple testing. All p-values are
reported in Supplemental Table S3 (available online at
We also performed one-tailed t-tests at all time points
between specific treatment groups to indicate the times at
which specific effects occur (Supplemental Fig. S2, available
online at www.liebertonline.com/ten).
SFRP4 REDUCES SCAR SIZE AFTER ISCHEMIC INJURY3331
Fibrosis and heart wall thickness
Wall thickness and fibrosis percentage mean values for
individual treatment groups were analyzed by a one-way
multivariate ANOVA with treatment groups specified as
either control (PBS or empty polyhedra delivered as de-
scribed) or Sfrp4 treated (soluble, or in polyhedra). The pa-
rameters of the negative linear relationship between fibrosis
percentage and wall thickness were estimated using the R
In vivo Sfrp4 release
Due to the fragmented nature of the Sfrp4 release data, we
performed a number of different univariate ANOVA analy-
ses on specific subsets of the data to ask the pertinent
questions (see Supplemental Fig. S3 for details, available
online at www.liebertonline.com/ten). Although this could
lead to the appearance of type I errors, the p-values obtained
were sufficiently small to pass Bonferroni corrections.
Gene expression, cell proliferation,
and vascular densities
These data sets were tested using paired t-tests as each one
contained only one reasonable control versus experimental
Sfrp4 transcription is upregulated
in ischemic heart models
We first examined the expression patterns of rat Sfrp4, b-
catenin, and collagen type IIIa in the ischemic border region of
LAD-ligated rat hearts using qRT-PCR to confirm expression
of these genes during wound healing (Fig. 1). As shown
in Figure 1, the levels of Sfrp4 transcripts were upregulated
in rat ischemic heart, whereas expression of b-catenin was not
markedly upregulated. The level of collagen IIIa transcripts
remained elevated throughout the 6 weeks examined, sug-
gesting an active tissue remodeling process that takes more
than a month after heart ischemic injury.
IM administration of Sfrp4 recombinant protein
improved cardiac function of ischemic heart
Sfrp4 recombinant protein was administered to the intra-
cardiac musculature just after ischemic injury to evaluate the
efficacy of administration of the agent in the acute phase.
Further deterioration of cardiac function as determined by
EF, FS (functional shortening), or fractional area change in
the LAD ligation model (Supplemental Table S4, available
online at www.liebertonline.com/ten) was blocked as a re-
pre 357 14 212842
pre 357 1421 28 42
pre 357 14 21 2842
nonischemic) areas were prepared at, before (pre), or 3, 5, 7, 14, 21, 28, and 42 days after LAD ligation. Gene expression of Sfrp4,
collagen type IIIa, or b-catenin was determined by qRT-PCR. The points and error bars show mean and standard deviations,
respectively, of four rat heart samples obtained at the designated times. FI stands for fold induction of qRT-PCR values at
designated times compared with that at pre. Sfrp4, secreted frizzled related protein 4; LAD, lateral anterior descendant; qRT-
Gene expression profile of ischemic-injury-related molecules. Heart tissues from nonischemic and border (ischemic/
termined by EF before and after ischemic or recanalization injuries. EFs were monitored by echocardiography at the indicated
time points and calculated as described in the Materials and Methods section. Other cardiac parameters, including fractional
shortening (FS) and functional area change, are shown in Supplemental Table S4. (A) IM injection of Sfrp4 in an acute
ischemic model. Ischemic hearts were generated by LAD ligation, and 100mL of PBS (n¼7), 5mg Sfrp4 protein (n¼6), or
20mg Sfrp4 protein (n¼9) was injected to the ischemic border zones soon after LAD ligation. (B) IM injection of Sfrp4 in a
subacute ischemic model. About 100mL of PBS (n¼5) or 20mg Sfrp4 protein (n¼6) was administered intramuscularly 2
weeks after LAD ligation. (C, D) IM injection of Sfrp4 just after (C) or before (D) recanalization injury. About 100mL PBS or
20mg Sfrp4 protein was administered intramuscularly after or before a transient 1h LAD ligation. All treatments except 5mg
Sfrp4 (A) show either significant (p<0.05) treatment or time-treatment interaction effects as judged by repeated measures
ANOVA, and the effect of 20mg Sfrp4 is different to that of 5mg (p¼2.38E-4). Summary measurements are shown in
Supplemental Fig. S1 and p-values are reported in Supplemental Table S3. PBS, phosphate-buffered saline; EF, ejection
fraction; IM, intramuscular; ANOVA, analysis of variance. Color images available online at www.liebertonline.com/ten.
Sfrp4 recombinant protein exerts a cardio-protective effect. Cardiac function of PBS- or Sfrp4-treated rats as de-
3332 MATSUSHIMA ET AL.
LAD ligation& injection
PBS sfrp4(5) sfrp4(20)
PBS sfrp4(5) sfrp4(20)
PBS sfrp4(5) sfrp4(20)
PBS sfrp4(5) sfrp4(20)
PBS sfrp4(5) sfrp4(20)
Intra-muscular injection of Sfrp4 in an acute ischemic model.
sfrp4(5) Sfrp4 protein 5mg treated,
sfrp4(20) Sfrp4 protein 20mg treated,
PBS sfrp4 PBS sfrp4PBS sfrp4
Intra-muscle injection of sfrp4 in a sub-acute ischemic model
sfrp4:Sfrp4 protein 20mg treated, n=6
Sfrp4 protein-treated, n=7
PBS sfrp4 PBS sfrp4PBS sfrp4
PBS sfrp4 PBS sfrp4PBS sfrp4
Intra-musclular injection of Sfrp4 in a reperfusion injury model
sfrp4:Sfrp4 protein-treated, n=6
Intra-musclular injection of Sfrp4 in a reperfusion injury model
sult of administrating Sfrp4 protein just after ischemic injury
in a dose-dependent manner (Fig. 2A).
We then examined the efficacy of Sfrp4 administration
during the subacute phase (2 weeks after LAD ligation). In-
terestingly, IM Sfrp4 protein injection resulted in a partial
recovery of cardiac function during the 3–10 weeks exam-
ined (Fig. 2B), suggesting that the tissue remodeling process
continues for at least several weeks after the ischemic injury.
Third, the therapeutic efficacy of Sfrp4 protein administra-
tion for the treatment of transient LAD ischemic attack was
evaluated using a rat LAD recanalization model. Similarly,
IM administration of 20mg Sfrp4 protein both before and
after LAD ligation improved cardiac function after reperfu-
sion injury (Fig. 2C, D).
Sfrp4 in a slow-releasing polyhedra form has a longer
and superior cardio-protective effect
As demonstrated in Figure 1, scar formation and the tissue
remodeling process takes more than a month. Hence, we
reasoned that administration of Sfrp4 in a long-lasting, slow-
releasing form might exert a stronger therapeutic effect on
ischemic heart recovery than simple injection. To examine
this, we used polyhedron cubes to facilitate a slow release of
Sfrp4. (Fig. 3A).23Release of the cargo protein from polyhe-
dra cubes occurs as a result of the decay of the polyhedra
cubes by proteases secreted from adjacent cells. Although we
have not determined the optimum therapeutic dose for S-PH
for this application, 5?103cubes of S-PH emit 1.6ng of Sfrp4
protein when incubated in the presence of primary rat car-
diomyocytes for 4 days (Fig. 3B) in 500mL culture medium,
and we estimated that 2.5?106polyhedra contain the
equivalent of 20mg of Sfrp4 protein (methods). We found
that 2.5?106cubes of S-PH block the deterioration of cardiac
function when administered intramuscularly at the onset of
ischemic injury, but we failed to detect any effect at a dose of
2.5?105cubes (data not shown). Indeed, statistical analysis
indicates that 20mg of soluble Sfrp4 shows the strongest ef-
fect at around 8 weeks, but the beneficial effect drops rapidly
after this time point (Fig. 3C; Supplemental Figs. S1 and S2).
On the other hand, administration of 2.5?106cubes of S-PH
provided a longer therapeutic effect than that of 20mg of
Sfrp4 recombinant protein especially at 8–20 weeks after
LAD ligation, although its effect also appears to drop after
8 weeks (Fig. 3C; Supplemental Figs. S1 and S2).
Schema for generation of empty polyhedra and Sfrp4 immobilized in polyhedra (S-PH) and scanning electron microscopic
image of S-PH. (B) Concentration of Sfrp4 in the culture medium after culture of primary rat cardiomyocytes in the presence
of S-PH (n¼3) for 2 or 4 days as determined by ELISA. Mean values and standard deviations are indicated. (C) Twenty
microliter PBS (n¼5), 2.5?106cubes of empty polyhedra (n¼6), 20mg Sfrp4 protein (n¼5), or 2.5?106cubes of S-PH (n¼6)
were administered just after LAD ligation. EFs of indicated groups are shown. The full data set contains significant treatment
effects as determined by repeated measures ANOVA (p¼1.1e-4). Significant (p¼0.016) time–treatment interaction effects
were also observed between polyhedra bound and soluble Sfrp4. Complete set of p-values are shown in Supplemental Table
S3. S-PH, Sfrp4 containing polyhedra. Color images available online at www.liebertonline.com/ten.
Administration of Sfrp4 in a slow-releasing cube polyhedra form demonstrated long-term therapeutic effects. (A)
3334 MATSUSHIMA ET AL.
ELISA measurements of serum Sfrp4 concentrations up to
10 weeks after Sfrp4 administration indicated small increases
in Sfrp4 levels that appear to be approximately correlated
with the efficacy of the treatment methods (Supplemental
S-PH can be applied in combination
with a biodegradable vehicle
The size of the rat heart makes it feasible to administer
therapeutic agents by IM injection; however, the large size of
the human heart may make it difficult to evenly distribute
Sfrp4 protein across the ischemic region by simple injection.
Collagen sheets have previously been used to facilitate
wound healing after serious burn and other types of skin
injury.24Collagen sheets show low immunoreactivity and
are degraded in tissues after a period of months. Since
polyhedra are resistant to dessication they can easily be
immobilized onto the surface of collagen sheets, and this
might provide a means to attain a uniformly localized and
enduring drug application to a large injured area. S-PH im-
mobilized on collagen type I sheets were applied to ischemic
areas of the heart (Fig. 4A, B) just after LAD ligation. This
intervention provided a similar level of protection against
heart dysfunction to that seen by straight injection of poly-
hedra (Fig. 4C). Histological examination showed neither
collagen type I sheet nor polyhedra cubes in heart sections 10
weeks after LAD ligation (data not shown), suggesting that
both the collagen sheet and polyhedra were absorbed in the
Alternatively, fibrin glue might provide a useful means of
applying polyhedra to larger ischemic areas. Fibrin glue is
presently used in a number of surgical applications and as
such has been validated for clinical use.25Hence, we also
tried plastering S-PH in fibrin glue to ischemic hearts 2
weeks after LAD ligation, as a model for the treatment of
patients in the subacute phase who have not received treat-
ment at the onset of ischemic attack. S-PH in fibrin glue
(Boveal) were plastered precisely to the ischemic heart area
following a reopening of the chest cavity 2 weeks after LAD
Schema for application of collagen sheet to ischemic left ventricle after LAD ligation. (B) Application of collagen sheet during
operation. (C) About 5.0?106cubes of empty polyhedra (n¼5) or S-PH (n¼6) were immobilized on collagen type I sheets by
air-drying and applied to the ischemic region of rat hearts just after LAD ligation. (D) About 5.0?106cubes of empty
polyhedra (n¼6) or S-PHs (n¼6) were mixed with 150mL of fibrin glue and plastered to ischemic regions of rat hearts 2
weeks after LAD ligation by reopening of the chest. EFs of respective group at designated time points are shown in the
graphs. The use of both collagen sheet (p¼2.96e-3) and fibrin glue (p¼3.5e-3) provided statistically significant effects as
judged by repeated measures ANOVA.
Sfrp4 polyhedra applied with a range of vehicles ameliorated cardiac dysfunction caused by ischemic injury. (A)
SFRP4 REDUCES SCAR SIZE AFTER ISCHEMIC INJURY3335
ligation. Plastering 5.0?106cubes of S-PH after 2 weeks of
infarction provided effective for the treatment of subacute
ischemic injury (Fig. 4D). This treatment also resulted in an
initial recovery of heart function in the first 2 weeks after
application in a similar manner to that seen for soluble Sfrp4
Sfrp4 attenuates the formation of acellular fibrous tissue
in the ischemic heart
Masson’s Trichrome staining of ischemic heart cross sec-
tions revealed that administration of Sfrp4 limited the
amount of acellular scar formed (Fig. 5A–D). The thicknesses
ligation were sectioned and stained by Masson’s Trichrome 10 weeks after LAD ligation. (B) Twenty-microgram Sfrp4
protein-injected heart, (C) 2.5?106cubes of S-PH injected heart, (D) 5?106cubes of S-PH immobilized collagen sheet-applied
heart, (E) nontreated ischemic heart section, (F) PBS-injected heart, (G) 2.5?106cubes of empty polyhedra-injected heart, (H)
5?106cubes of empty polyhedra-immobilized collagen sheet-applied heart. Photo of representative cross section of each
group (n¼4) is shown. (I) Means of fibrosis percentage and wall thickness for individual treatment groups plotted against
each other. Vertical and horizontal lines indicate standard errors of mean measurements for fibrosis and wall thickness,
respectively; the diagonal line indicates the line of best fit. IM injection of PBS or Sfrp4; PH IM injection of empty or S-PH; PH-
sheet, application of empty or S-PH immobilized on collagen sheet; PH-glue, empty or S-PH applied with fibrin glue. Filled
circles indicate control treatments; empty circles indicate Sfrp4-containing treatments. Sfrp4 treatments result in hearts with
significantly less fibrosis and thicker walls (p¼8.4e-4) as indicated by multivariate ANOVA.
Histology of PBS- or Sfrp4-treated hearts. Adult normal rat heart (A) and ischemic hearts (B–H) generated by LAD
3336MATSUSHIMA ET AL.
of the LV walls were reduced, whereas the size of the left
ventricle cavities were markedly enlarged in non- or PBS-
treated ischemic hearts (Fig. 5E–H). This suggests that these
rat hearts underwent chronic heart failure beyond compen-
satory pump mechanism 10 weeks after ischemic injury. In
contrast, the sizes of the left ventricle cavities were not en-
larged in Sfrp4-treated hearts.
We performed semi-quantitative measurements of wall
thickness and the percent of fibrosis in heart sections indi-
cated above. This analysis revealed a surprisingly strict
negative linear relationship between wall thickness and fi-
brosis percentage (Fig. 5I). Multivariate ANOVA analysis of
the mean values for the individual treatment groups indi-
cated a highly significant effect for Sfrp4 application (p¼
8.5e-4). However, the data do not allow us to determine any
differences in the effect for the individual Sfrp4 treatment
To investigate the cellular effect of Sfrp4 administration on
ischemic injury, LAD-ligated heart sections were stained
with anti-collagen type III- and anti-cTn-T-antibody. The
number of cardiomyocytes was drastically reduced; instead,
large numbers of cells lacking cTn-T but producing collagen
III were present in the interstitial region after ischemic injury
in both control and Sfrp4-treated hearts. However, PBS-
treated hearts were characterized by the presence of a
large acellular fibrous scar as evidenced by the lack of 40,6-
diamidino-2-phenylindole (DAPI) and cTn-T staining (Fig.
6A, white arrow). This acellular scarring was markedly
suppressed in Sfrp4-treated hearts where it was replaced
with cellular, but noncardiac, tissue (Fig. 6A). Administra-
tion of 2.5?106cubes of S-PH generated similar histological
features to that of 20mg of Sfrp4 protein (data not shown).
Administration of Sfrp4 attenuated the activation
PBS- or Sfrp4-treated ischemic hearts 3 days after LAD
ligation were stained for b-catenin and Sfrp4 using a series of
antibodies. Interestingly, the acellular scar (evidenced by the
lack of DAPI and cTn-T staining) was already present 3 days
after ischemic injury (Fig. 6B). b-catenin is an intracellular
protein known to mediate two distinct biological signaling
mechanisms: the Wnt canonical pathway and cell–cell ad-
hesion through interactions with cadherins.26,27The majority
of intracellular b-catenin resides as a membrane-associated
form in nonischemic heart regions (Fig. 6B). However, this
intracellular localization of b-catenin was disturbed upon
ischemic injury, suggesting that signaling associated with the
relocation of b-catenin was triggered upon ischemic injury.
GSK-3b, which phosphorylates and marks b-catenin for deg-
radation, was inactivated upon ischemic injury as indicated
by the detection of the inactive Serine 9-phosphorylated
GSK-3b form (Fig. 6D). Inactive Serine 9-phosphorylated
GSK-3b, activated b-catenin (Fig. 6C), and BrdU incorpora-
tion (Fig. 7A) were detected selectively at the densely pop-
ulated ischemic border area (shown by nuclear staining;
Supplemental Fig. S4, available online at www.liebertonline
.com/ten). Although we failed to show at the individual cell
level that the presence of active b-catenin correlates with cell
proliferation (due to the use of DAB detection for BrdU
staining) and production of collagen (due to using primary
antibodies of the same species), our observations suggest that
b-catenin activation stimulates the proliferation of collagen
producing cells at the ischemic border area. The upregulation
of the b-catenin effector TCF4,20,28its down stream target
gene cyclin D2, and collagen IIIa transcripts upon ischemic
injury (Fig. 7C) support this idea.
Interestingly, endogenous Sfrp4 was induced upon ische-
mic injury (Fig. 6B) in agreement with our qRT-PCR data
(Fig. 1), suggesting a physiological role for Sfrp4 in deter-
mining the scar size. Our data demonstrate that adminis-
tration of Sfrp4 at the onset of ischemic attack suppressed the
inactivation of GSK-3b (Fig. 6D), activation of b-catenin (Fig.
6C), cell proliferation (Fig. 7A), decrease in vascular density
(Fig. 7B), as well as induction of TCF4, cyclin D2, and collagen
IIIa transcription (Fig. 7C). These results suggest that this
further inhibition of b-catenin signaling is responsible for the
therapeutic effect of Sfrp4 administration.
In this work we demonstrate that the application of Sfrp4
protein to ischemic regions attenuates postinfarct degrada-
tion of heart function in two related rat ischemia models.
Further, we show that this effect can be prolonged by the
immobilization of Sfrp4 in insect polyhedra particles and
demonstrate means by which such particles might be applied
in a clinical setting. Although the beneficial effects of Sfrp4
application are limited, they are highly reproducible and can
be observed in both transient and permanent LAD ligation
models. Further, the extension of the duration of therapeutic
effect observed when Sfrp4 was applied in a polyhedra for-
mat is consistent with the extended period of elevated serum
Sfrp4 (Supplemental Fig. S3). Our data also suggest, but
cannot be used to argue, that the application of S-PH within
collagen sheets or fibrin glue may extend the therapeutic
effect for a longer period (Figs. 3C and 4E, F). However, this
might also be related to the higher dosage of polyhedra ap-
plied in these experiments. In any case we have not made
any efforts to determine the optimal dosage of Sfrp4 and it
seems likely that more careful titration of Sfrp4 dosage
would result in stronger effects.
This is not the first report to implicate the role of Sfrps in
the ischemic response; contrary to our observations, it has
previously been shown that overexpression of Sfrp1 in mice
can lead to an increase in infarct size.29Similarly, it has re-
cently been reported that loss of Sfrp2 in transgenic mice
results in less fibrosis of the heart after surgically induced
ischemia.30These apparently contradictory effects of Sfrps on
the pathology of ischemic heart injury might reflect struc-
tural differences within the Sfrp family of proteins that result
in different physiological effects.31Alternatively, the differ-
ent observations might result from differences in the animal
models used, as the transgenic models were, for example,
unable to limit the effect of Sfrp antagonism or over-
expression to only the ischemic injury site.
Role of active b-catenin and scar formation
in ischemic heart
Our findings indicate that ischemic injury triggers the
activation of Wnt signaling, resulting in cell proliferation and
the deposition of extracellular matrix in the ischemic area.
Sfrp4 expression is upregulated endogenously upon ische-
mic injury, suggesting that it acts to regulate extracellular
SFRP4 REDUCES SCAR SIZE AFTER ISCHEMIC INJURY3337
or 20mg Sfrp4 protein were sectioned 10 weeks after LAD ligation. Slides were costained with antibodies against collagen
type III (green) and cTn-T (red). The extent of the noncardiac acellular scar (lack of cTn-T and 40,6-diamidino-2-phenylindole
(DAPI) staining), indicated by white arrows, was suppressed by administration of Sfrp4. (B–D) About 100mL PBS or 20mg
Sfrp4 protein was injected into ischemic border areas just after LAD ligation to investigate the effects on Wnt canonical
signaling at early time points. Heart sections 3 days after LAD ligation were stained for b-catenin (green, B), Sfrp4 (red, B),
active b-catenin (green, C), and inactive serine 9 phosphorylated glycogen syntase kinase (GSK)-3b (red, D). DAPI (blue) was
used to observe nuclei and hence cellular versus acellular areas. The border between the acellular scar and cellular areas is
shown by dotted white lines. The average of total fibrous tissue areas (in mm2) detected by Masson’s Trichrome staining in
adjacent sections is indicated with standard deviations (C, D).
Administration of Sfrp4 interferes with Wnt canonical signaling. (A) Ischemic hearts treated by IM injection of PBS
3338 MATSUSHIMA ET AL.
matrix deposition by tempering Wnt canonical signaling at
the receptor level. Excessive deposition of extracellular ma-
trix may limit the entry and survival of cells in the ischemic
area and thus lead to the formation of acellular scarring.
Hence, the therapeutic effect of Sfrp4 administration may be
mediated through the promotion of the formation of a rela-
tively nonfibrous cardiac wall in the ischemic area.
Recently, TGF-b1 was reported to induce an endothelial to
mesenchymal transition (EndMT) in the mouse pressure
overloading model contributing to cardiac fibrosis. Systemic
administration of recombinant bone morphogenetic protein-
7 significantly inhibited the EndMT and the resulting pro-
gression of cardiac fibrosis.32Hence, at least two distinct
signal pathways leading to extracellular matrix deposition
could be employed depending on the type of heart tissue
injury. Cardiac fibrosis is coupled with long-term mechanical
stretching and is widely observed in chronic heart fail-
ure,33,34whereas ischemic heart injury requires prompt and
massive mesenchymal cell proliferation at the initial stage to
mitigate massive cell death resulting from anoxia. In this
context, activation of b-catenin resulting in cell proliferation
may be the major event of the tissue repair process after
Anti-fibrosis therapy by Sfrp4
The suppression of acellular scar formation in ischemic
hearts by Sfrp4 administration suggests a wider therapeutic
application of Sfrp4. Upregulation of Sfrp4 and Wnt agonists
has been observed both in the skin of systemic sclerosis pa-
tients35and during kidney fibrosis caused by unilateral uri-
nary tract obstruction.36Indeed, a therapeutic effect of Sfrp4
after LAD ligation. About 2.5?106cubes of empty polyhedra (empty-PH), or 2.5?106cubes of S-PH were injected into
ischemic border areas just after LAD ligation. BrdU was administered at day 3. The border between the acellular scar and
cellular areas is shown by dotted black lines. Cubes visible in the bottom of the photo are S-PH in the ischemic border area.
The mean and standard deviations of the number of BrdU-positive cells are indicated in the figure. (B) Vascular density in the
ischemic border area of PBS- or Sfrp4-treated hearts. The number of von Willebrand Factor-expressing vessel-like structures
in border areas of PBS- or Sfrp4-treated rats 3 days after LAD ligation. Means and standard deviations of vascular density
(vessels per mm2) are shown in the figure. Red arrows indicate von Willebrand Factor-positive vessel-like structures. Black
line framed area in upper photo is magnified and shown (indicated as block arrow) in lower photo. (C) Transcript levels of
the b-catenin effector gene TCF4, the TCF4 target cyclin D2, and collagen IIIa (Col IIIa) from PBS or 20mg Sfrp4-treated ischemic
border areas 3 days after ischemic heart injury as determined by qRT-PCR. Values were normalized by the expression of
glyceraldehyde 3-phosphate dehydrogenase (rat glyceraldehyde 3-phosphate dehydrogenase as 10,000). The bar and error
bar show mean and standard deviations of three rat heart samples, respectively. p-Values for differences in mean values for
PBS- or Sfrp4-treated tissues are indicated in the figure.
Sfrp4 suppresses cell proliferation and collagen production after ischemia. (A) BrdU incorporation during day 3 to 4
SFRP4 REDUCES SCAR SIZE AFTER ISCHEMIC INJURY3339
in the treatment of kidney fibrosis has been confirmed.36
Further, Dupuytren’s disease, a superficial fibromatosis of
the hand, has been reported to be caused by aberrant acti-
vation of b-catenin.37,38In these cases, tissue-engineered
scaffolds with S-PH may provide a stronger clinical effect by
providing a longer and precise topical drug delivery system
throughout the wound healing process.
We thank T. Yashita for excellent animal experiments, Y.
Miyamoto for tissue section stain work, A. Nagahashi for
technical assistance for RT-PCR, R. Hisamori for typing and
editorial help, Dr. David Smith for advice on statistical an-
alyses, and SI. Nishikawa, Y. Murakami, and H. Hirata for
valuable advice and discussion. This study was supported
by the grant from Ministry of Industry and Economy (2007–
20R5021 in Japan.
Innovation 21 project
No competing financial interests exist.
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Address correspondence to:
Shin Kawamata, M.D., Ph.D.
Basic Research Group for Regenerative Medicine
Foundation for Biomedical Research and Innovation
Hajime Mori, Ph.D.
Department of Applied Biology
Kyoto Institute of Technology
Received: November 16, 2009
Accepted: June 1, 2010
Online Publication Date: July 17, 2010
SFRP4 REDUCES SCAR SIZE AFTER ISCHEMIC INJURY3341
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