Stem Cell Research (2011) 6, 206–214

available at www.sciencedirect.com

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REGULAR ARTICLE

Human mesenchymal stem cell-conditioned

medium improves cardiac function following
myocardial infarction
Leo Timmers a , Sai Kiang Lim b,c,⁎, Imo E. Hoefer a , Fatih Arslan a ,
Ruenn Chai Lai b,d , Angelique A.M. van Oorschot e , Marie Jose Goumans e ,
Chaylendra Strijder a , Sui Kwan Sze f , Andree Choo g , Jan J. Piek h ,
Pieter A. Doevendans a , Gerard Pasterkamp a , Dominique P.V. de Kleijn a,i,⁎⁎
a
Department of Cardiology, University Medical Center Utrecht, Netherlands
b
Institute of Medical Biology, A*STAR, Singapore
c
Department of Surgery, YLL School of Medicine, NUS, Singapore
d
NUS Graduate School for Integrative Sciences & Engineering, NUS, Singapore
e
Department of Molecular Cell Biology, Leiden University Medical Center, Netherlands
f
Nanyang Technological University, Singapore
g
Biotechnology Institute, Singapore
h
Department of Cardiology, Academic Medical Center, Amsterdam, Netherlands
i
Interuniversity Cardiology Institute of the Netherlands, Utrecht, Netherlands

Received 21 August 2010; received in revised form 29 December 2010; accepted 3 January 2011
Available online 28 January 2011

Abstract Recent studies suggest that the therapeutic effects of stem cell transplantation following myocardial infarction (MI)
are mediated by paracrine factors. One of the main goals in the treatment of ischemic heart disease is to stimulate vascular
repair mechanisms. Here, we sought to explore the therapeutic angiogenic potential of mesenchymal stem cell (MSC)
secretions. Human MSC secretions were collected as conditioned medium (MSC-CM) using a clinically compliant protocol. Based
on proteomic and pathway analysis of MSC-CM, an in vitro assay of HUVEC spheroids was performed identifying the angiogenic
properties of MSC-CM. Subsequently, pigs were subjected to surgical left circumflex coronary artery ligation and randomized to
intravenous MSC-CM treatment or non-CM (NCM) treatment for 7 days. Three weeks after MI, myocardial capillary density was
higher in pigs treated with MSC-CM (645 ± 114 vs 981 ± 55 capillaries/mm2; P = 0.021), which was accompanied by reduced
myocardial infarct size and preserved systolic and diastolic performance. Intravenous MSC-CM treatment after myocardial
infarction increases capillary density and preserves cardiac function, probably by increasing myocardial perfusion.
© 2010 Elsevier B.V. All rights reserved.

⁎ Correspondence to: S.K. Lim, Institute of Medical Biology, A*STAR, Department of Surgery, YLL School of Medicine, NUS, 8A Biomedical
Grove, No. 05–505 Immunos, Singapore 136648. Fax: +65 6464 2048.
⁎⁎ Correspondence to: D.P.V. de Kleijn, Laboratory of Experimental Cardiology, University Medical Center Utrecht, Room G02.523,
Heidelberglaan 100, 3584 CX Utrecht, The Netherlands. Fax: +31 30 2522693.
E-mail addresses: saikiang.lim@imb.a-star.edu.sg (S.K. Lim), d.dekleijn@umcutrecht.nl (D.P.V. de Kleijn).

1873-5061/$ - see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.scr.2011.01.001

Introduction
Despite significant advances in myocardial revascularization,
coronary artery disease and subsequent myocardial infarc-
tion (MI) are leading causes of morbidity and mortality
worldwide. One of the main goals in the treatment of
ischemic heart disease is the development of effective
strategies to stimulate vascular repair mechanisms in order
to achieve adequate tissue perfusion.
Although the potential of stem cell therapy for MI is still
heavily debated as a result of studies that failed to show
improved outcome (Moelker et al., 2006; de Silva et al., 2008;
Janssens et al., 2006; Lunde et al., 2006; Meyer et al., 2009;
Menasche et al., 2008), there are several preclinical and
clinical studies that demonstrated the therapeutic effects of
stem cell transplantation (Hashemi et al., 2008; Wolf et al.,
2009; Assmus et al., 2006; Schachinger et al., 2006; Strauer
et al., 2002; Yousef et al., 2009). While robust evidence for
cardiomyocyte replacement is still limited, more evidence is
accumulating for vascular repair mechanisms following stem
cell transplantation (van Laake et al., 2009; Losordo &
Dimmeler, 2004; Erbs et al., 2007). Among the stem cells that
have been tested for cardiac repair, mesenchymal stem cells
(MSCs) derived from adult bone marrow have emerged as a
promising stem cell type for treating cardiovascular disease
(Pittenger & Martin, 2004). Recent studies suggest that at
least some of the therapeutic effects of MSCs are mediated by
paracrine factors secreted by the cells (Caplan & Dennis,
2006; Gnecchi et al., 2005). These paracrine factors could be
exploited to extend the therapeutic possibilities of MSCs for
the treatment of a variety of diseases, including MI.
Application of MSC secretions rather than MSCs themselves
in order to restore tissue perfusion could enable us to avoid
some of the limiting factors associated with cell therapy, such
as immune incompatibility, tumorigenicity, costs, and wait-
ing time for ex vivo expansion.
We previously described how human MSC lines can be
reproducibly generated from human embryonic stem cell
(hESC) lines, which constitute an invariable source of
consistently uniform batches of MSCs (Lian et al., 2007). In
addition, we established for the first time the collection of
these MSC secretions as conditioned medium (MSC-CM) in a
clinically compliant manner, circumventing exposure to
virus, mouse cells, or serum (Sze et al., 2007). Recently, we
demonstrated that MSC-CM treatment following ischemia/
reperfusion injury reduces myocardial apoptosis and oxida-
tive stress (Timmers et al., 2007a). In the current paper, we
reveal the angiogenic potential of clinically compatible MSC-
CM and demonstrate in a large animal model that it can be
used to improve cardiac function following MI.
Results
MSC-CM stimulates angiogenesis in vitro
Ingenuity pathway analysis of the secretory proteome of
MSC-CM revealed that within the physiological system
development and function group, cardiovascular system
development and function has the highest overrepresenta-
tion (P b 10-12) with 75 different focus proteins. Within the
group of cardiovascular system development and function,
207
development of blood vessels, angiogenesis, proliferation of
endothelial cells, neovascularization, and vascularization
are the processes with the highest overrepresentation
(supplementary data, Table 1).
Having identified the potential involvement of MSC-CM in
blood vessel formation, we used an in vitro assay of
angiogenesis to further investigate this potential. In an
angiogenesis assay based on formation of capillary sprouts by
HUVEC spheroids, MSC-CM induced significantly more capil-
lary sprouting compared with NCM (Fig. 1A). The average
length of the sprouts did not increase following MSC-CM
treatment compared with NCM (Fig. 1B). The total length of
all sprouts, however, significantly increased in spheroids
incubated with MSC-CM when compared with NCM (Fig. 1C).
In addition, a HUVEC/Matrigel angiogenesis assay was
performed, which revealed a longer average tube length with
CM compared with NCM (1.07 ± 0.046 vs 0.99 ± 0.041 mm;
P = 0.003).
MSC-CM treatment after infarction increases
capillary density and decreases collagen density
Capillary density was higher in the border areas following
MSC-CM treatment compared with NCM treatment (Figs. 2A
and B). In the remote area, capillary density did not differ
between MSC-CM treatment and NCM treatment (Fig. 2C).
We were unable to reliably quantify capillary density in the
infarct area due to the unorganized structure of the infarct
granulation tissue 3 weeks following MI.
Picrosirius staining revealed that collagen density in the
border area and in the remote myocardium was lower in pigs
treated with MSC-CM compared with pigs treated with NCM.
In the infarct area, no difference was detected between the
treatment groups (Fig. 3).
MSC-CM preserves cardiac function following MI
One hour after coronary artery ligation, cardiac function
decreased similarly in both groups. Fractional area shorten-
ing decreased from 45.8 ± 1.9 to 32.3 ± 2.0% in the NCM group
and from 46.9 ± 4.6 to 29.7 ± 3.4% in the MSC-CM group
(P = 0.670). Also cardiac output (2.80 ± 0.10 L/min (NCM) vs
2.67 ± 0.22 L/min (MSC-CM); P = 0.596), mean aortic pressure
(90 ± 8.3 mm Hg (NCM) vs 84 ± 5.4 mm Hg (MSC-CM); P = 0.544)
and dP/dtMAX (1135 ± 83 mm Hg/s (NCM) vs 1096 ± 83 mm Hg/s
(MSC-CM); P = 0.741) were similar in both groups 1 h after
coronary artery ligation.
In the 3 weeks following MI, the LV internal areas
increased in all pigs. However, the increase in LV internal
area tended to be more pronounced in NCM-treated animals,
pointing to reduced remodeling in MSC-CM-treated pigs
(Figs. 4A and B). Echocardiographic wall thickness of the
infarct area decreased in pigs treated with NCM, but not in
pigs treated with MSC-CM (Fig. 4C). The fractional area
shortening was higher following MSC-CM treatment, which
reflects increased systolic cardiac performance (Fig. 4D).
Also other systolic functional parameters such as ejection
fraction, dP/dtMAX, stroke volume, and stroke work were
higher in animals treated with MSC-CM. Improved cardiac
function translated into improved hemodynamic parameters,
with the mean arterial pressure and cardiac output being
208 L. Timmers et al.

Figure 1 HUVEC spheroid assay of angiogenesis. The number of capillary sprouts (A), mean length of sprouts (pixels, B), and total
length of sprouts (pixels, C) after incubation of HUVEC spheroids with MSC-CM (n = 12) or NCM (n = 12). Representative pictures of MSC-
CM and NCM are demonstrated in panels D and E, respectively. * P b 0.01 vs NCM.

higher in MSC-CM-treated pigs. In addition, diastolic function
improved, as assessed with dP/dtMIN and diastolic chamber
stiffness. All functional parameters are summarized in Table 1.
Myocardial infarct size 3 weeks following coronary artery
ligation was smaller in MSC-CM-treated pigs compared to NCM-
treated pigs (11.6 ± 2.0% vs 16.6 ± 1.2% of the LV; P = 0.050).
Two pigs died during the surgical procedure due to
refractory ventricular fibrillation before treatment with
MSC-CM or NCM, and were therefore excluded from the
study. Two pigs (1 treated with MSC-CM and 1 treated with
NCM) died in the third week following MI without signs of
heart failure or cardiac rupture, probably due to ventricular
tachycardia/fibrillation.
Discussion
Here, we demonstrate that human MSC-CM treatment
enhances capillary density, resulting in reduced myocardial
infarct size and preserved systolic and diastolic function in a
porcine model of myocardial infarction.
Recently, it was demonstrated that administration of MSC
secretions improved outcome in a murine model of MI
(Gnecchi et al., 2005). Vascular repair mechanisms consti-
tute an important means to reduce myocardial injury and to
improve cardiac function following MI. Although the thera-
peutic potential of stem cell transplantation is still a subject
of discussion, evidence for vascular repair mechanisms
following stem cell transplantation is growing (van Laake
et al., 2009; Losordo & Dimmeler, 2004; Erbs et al., 2007). If
vascular repair mechanisms are induced by paracrine
factors, application of stem cell secretions may extend the
repertoire of stem cell-based therapies with a different
dimension.
An in vitro sprouting assay confirmed Ingenuity analysis of
earlier MSC-CM proteomics data and identified the angio-
genic potential of MSC-CM. In order to investigate the
therapeutic potential of MSC-CM to stimulate angiogenesis

Figure 2 Capillary density. Representative sections of border areas after lectin staining to visualize endothelium, 3 weeks after MI,
from pigs treated with NCM (A, n = 9) or MSC-CM (B, n = 9). Quantification of capillary density in remote and border area is
demonstrated in panel C. Scale bars, 500 μm.
Figure 3 Collagen density. Quantification of collaged density
in the infarct, border, and remote areas in pigs treated with NCM
and MSC-CM.
209
in vivo, we used a porcine model of myocardial infarction.
During myocardial ischemia following coronary artery liga-
tion, cardiomyocytes in the central infarct perish within
minutes to hours after coronary occlusion. The border zone
of the ischemic area, however, contains injured but viable
cells under hypoperfused and hypoxic conditions which may
either be salvaged or die, depending on the conditions after
ischemia. Restoration of myocardial perfusion by means of
angiogenesis restores the metabolic needs of cardiomyocytes
in the border area and offers the cells a better chance of
survival, which in turn could lead to reduced myocardial
injury. Intracoronary delivery of bone marrow-derived
progenitor cells has been suggested to promote vascular
repair following MI, potentially via paracrine mechanisms
(Erbs et al., 2007). Such effect was also observed in a rodent
model of hind limb ischemia (Kinnaird et al., 2004). As
became evident from our histological analyses, MSC-CM
increased capillary density in the border areas and myocar-
dial infarct size was reduced, which was accompanied by
preserved LV dimensions and cardiac function.
Besides stimulation of angiogenesis, it cannot be excluded
that also other mechanisms contributed to reduction of
210 L. Timmers et al.

A B

LVES area (cm2)

LVES area (cm2)

*

C D

*
WT infarct (cm)

FAS (%)
E F
*
dP/dtMAX (mmHg/s)

dP/dtMin (mmHg/s)

Figure 4 Cardiac function. Left ventricular (LV) end-diastolic internal area (A), LV end-systolic internal area (B), posterolateral
myocardial wall (infarct) thickness (C), fractional area shortening (FAS, D), dPdtMAX (E), and dPdtMIN (F), before MI (baseline), 1 h post
MI and 3 weeks post MI in pigs treated with MSC-CM (black dots, n = 9) or NCM (open dots, n = 9). * P b 0.05 vs NCM.

myocardial infarct size and preservation of myocardial
function. With preserved left ventricular dimensions and
infarct thickness, inhibition of post MI cardiac remodeling is a
potential contributing mechanism. Collagen density in the
border area and remote myocardium was lower in pigs
treated with MSC-CM. Cardiac fibrosis is associated with
heart failure following myocardial infarction and may
contribute to progression of systolic and diastolic dysfunc-
tion (Burlew & Weber, 2002; Cleutjens et al., 1999). We
recently demonstrated that intravenous and intracoronary
MSC-CM treatment reduces myocardial apoptosis and oxida-
tive stress (Timmers et al., 2007a). In that study, cardio-
myocyte death was targeted acutely following ischemia and
reperfusion injury. In the current study, treatment was
started late after coronary artery ligation, and a model of
permanent coronary artery ligation was used in order to
investigate the angiogenic potential of MSC-CM and its effect
on cardiac function independent of reduction of reperfusion
injury. The fact that cardiac function improved using this
protocol shows that there may be additional benefit from
prolonged treatment with MSC secretions.
For this study, a permanent coronary artery ligation was
performed in anesthetized animals and functional measure-
ments were performed with open chest. In addition, we have
not been able to investigate the effect of common
comorbidities such as hypertension, dyslipidemia, and
diabetes, which might influence the biologic effect of MSC
secretions. Due to the permanent coronary artery ligation,
we were not able to assess the area at risk 3 weeks post MI.
We were not able to oversee the effects of human protein
injection on the immune system of the porcine host and the
potential influence on study endpoints. Furthermore, the
molecular mechanism for the induction of angiogenesis and/
or arteriogenesis and an exact determination of the
responsible angiogenic factors are still lacking. Several
clinical trials with potent angiogenic factors failed to
improve outcome in patients (Henry et al., 2003; Simons
et al., 2002). It is not clear how paracrine factors in stem cell
transplants, or the paracrine factors alone, can induce a
better effect. Most likely, a combination of angiogenic
factors rather than a single protein is responsible. Also
exosomes could play an important role. We previously
demonstrated exosomes in MSC-CM to contain and deliver
cardioprotective compounds preventing cardiomyocyte
death following ischemia and reperfusion injury (Lai et al.,
2010). Exosomes constitute a potential delivery tool to
 211

Table 1 Hemodynamic and Functional Parameters.

Baseline 3 weeks post MI Change from baseline (%)
Parameter NCM CM NCM CM NCM CM
HR, bpm 66 ± 4 73 ± 6 84 ± 6 86 ± 6 28 ± 12 12 ± 11
MAP, mm Hg 95 ± 5 89 ± 5 66 ± 6 85 ± 5 * -30 ± 5 -5 ± 8 *
CO, L/min 3.1 ± 0.1 3.0 ± 0.2 3.7 ± 0.6 5.3 ± 0.5 23 ± 21 78 ± 20
dP/dt max, mm Hg/s 1213 ± 77 1241 ± 56 1157 ± 162 1650 ± 124 * 4 ± 13 40 ± 9 *
dP/dt min, mm Hg/s -1274 ± 122 -1130 ± 96 -883 ± 98 -1313 ± 95 * 27 ± 9 -19 ± 19 *
EDP, mm Hg 8.0 ± 0.3 7.1 ± 0.6 13.3 ± 1.6 12.1 ± 1.1 68 ± 21 94 ± 42
WT infarct, cm 0.79 ± 0.04 0.83 ± 0.05 0.55 ± 0.02 0.79 ± 0.05 * -27 ± 4 -5 ± 4 *
Lvia (ED), cm2 15.8 ± 0.5 14.2 ± 1.0 26.0 ± 1.8 21.8 ± 1.5 64 ± 12 55 ± 12
Lvia (ES), cm2 8.5 ± 0.4 7.6 ± 0.9 17.7 ± 1.6 12.5 ± 0.9 * 104 ± 14 60 ± 14 *
FAS, % 45.8 ± 1.9 46.9 ± 4.6 32.6 ± 2.2 42.4 ± 3.3 * -28 ± 6 -6 ± 8 *
SV, ml 48 ± 3 42 ± 2 43 ± 4 62 ± 3 * -4 ± 11 52 ± 10 *
EDV, ml 134 ± 16 129 ± 9
ESV, ml 93 ± 15 73 ± 9
EF, % 33.7 ± 4.4 49.1 ± 3.3 *
SW, mm Hg·ml 2497 ± 333 4595 ± 361 *
Stiffness, mm Hg/ml 0.26 ± 0.04 0.14 ± 0.02 *
Summary of cardiac functional parameters of pigs treated with NCM (n = 9) and MSC-CM (n = 9), determined with echocardiography and
conductance catheter-based LV pressure and volume measurements. MI indicates myocardial infarction; HR, heart rate; MAP, mean arterial
pressure; CO, cardiac output; EDP, end-diastolic pressure; WT, wall thickness; LVia, left ventricular internal area; SV, stroke volume; EDV,
end-diastolic volume; ESV, end-systolic volume; EF, ejection fraction; SW, stroke work; Stiffness, end-diastolic chamber stiffness. * P b 0.05
vs NCM.

effectively carry angiogenic factors into the intracellular
space of the target tissue, protecting the factors from
proteases. This may explain why relatively low doses can
have such profound angiogenic effects. Whether exosomes
and their content are also responsible for the stimulation of
angiogenesis following MI remains a subject for further
investigation.
Here, we demonstrate that human MSC secretions
stimulate angiogenesis. These data support the hypothesis
that MSC transplantation induces vascular repair mechanisms
via paracrine pathways. Most importantly, human MSC
secretions harvested as conditioned medium using a clini-
cally compliant protocol reduced infarct size and preserved
cardiac function in a large animal model of myocardial
infarction. The study therefore identifies MSC-CM as a novel
therapeutic biologic in the improvement of cardiac function
after myocardial infarction.
expression profile, were generated by trypsinization and
propagation of hESCs from either the HuES9 hESC line or the
H1 hESC line in feeder- and serum-free selection media (Lian
et al., 2007). One of these cultures, HuES9.E1, can be stably
expanded for at least 80 population doublings. To harvest MSC
secretions, hESC-derived MSC cultures were transferred to a
chemically defined, serum-free culture medium to condition
the medium for 3 days before the media containing MSC
secretions were collected, clarified by centrifugation, con-
centrated 25 times using 10-kDa MW cutoff ultrafiltration
membranes and sterilized by filtration through a 220-nm
filter. The protein concentration after concentration was
0.50 mg/ml. As a negative control, the above-noted serum-
free culture medium was processed equally (nonconditioned
medium [NCM]).
Ingenuity pathway analysis

Methods For this analysis we used the proteomic data obtained

MSC-CM preparation
The protocols for MSC generation and CM preparation have
been described in previous papers (Lian et al., 2007; Sze et al.,
2007). In short, a chemically defined serum-free culture
medium (DMEM without phenol red, supplemented with insulin,
transferrin, and selenium, 5 ng/ml FGF2, 5 ng/ml PDGF AB,
glutamine-penicillin-streptomycin, and ß-mercaptoethanol)
was conditioned by MSCs derived from human embryonic
stem cells (hESCs) using a clinically compliant protocol. Three
polyclonal, karyotypically stable, and phenotypically MSC-like
cultures, that do not express pluripotency-associated markers
but displayed MSC-like surface antigens (CD29+, CD44+,
CD49a+/e+, CD105+, CD166+, CD34–, CD45–) and gene
previously (Sze et al., 2007). Bioinformatic analysis was
performed using Ingenuity pathway analysis (Ingenuity IPA-
2002 version 7.1). The 201 proteins were uploaded in
Ingenuity with Ingenuity Knowledge Base as a reference set
and direct and indirect relationships were included. All
molecules or relationships were considered for the filter
summary. In the summary, the focus was on Top Bio functions
in which physiological system development and function was
analyzed in depth.
HUVEC spheroid assay
Spheroids were formed from HUVECs to determine the effect
of conditioned medium on angiogenic sprout formation (Korff
& Augustin, 1998). Spheroids were formed overnight in 20%
212
methocel in culture medium and subsequently embedded in
a collagen–methocel matrix. Sprouting was induced over-
night with MSC-CM (n = 12) or non-CM (NCM) (n = 12) to
determine the effect of the secreted chemotactic proteins
on capillary sprouting. After overnight sprouting, spheroids
were fixed in 10% formaldehyde and pictures were taken and
the length and number of sprouts were quantified.
Animals
All experiments were performed in accordance with the
“Guide for the Care and Use of Laboratory Animals” prepared
by the Institute of Laboratory Animal Resources and with prior
approval by the Animal Experimentation Committee of the
Faculty of Medicine, Utrecht University, the Netherlands.
Sample size calculation
In a previous study, the mean fractional area shortening
(FAS), after LCx ligation was 36.5% (Timmers et al., 2007b).
With an alpha of 0.05, power of 0.80, SD of 5.8, and
difference of 20% considered relevant, 10 animals per group
were needed. To allow for 10% mortality, 11 animals per
group were enrolled.
Pig study design
Myocardial infarction was induced in 22 Dalland Landrace
pigs (60–70 kg, IDDLO, Lelystad, the Netherlands) by surgical
left circumflex coronary artery (LCx) ligation. Two pigs died
perioperatively before randomization and were therefore
excluded from the study. The remaining 20 pigs were
randomized after surgery by using sealed envelopes to
intravenous treatment with MSC-CM (2.0 ml MSC-CM, i.e.,
1.0 mg protein) or 2.0 ml NCM, initiated 4 h after coronary
artery ligation and the treatment was continued for 7 days
twice daily via a catheter inserted into the jugular vein. The
pigs were sacrificed 3 weeks after MI. To prevent thrombotic
complications and arrhythmias, all pigs were treated with
clopidogrel 75 mg/day from 3 days before MI until termina-
tion and amiodarone 400 mg/day from 10 days before MI
until termination.
Myocardial infarction and operational procedure
Pigs were anesthetized as described previously (Timmers et
al., 2007b). During the entire operation, electrocardiogram,
arterial pressure, and capnogram were continuously moni-
tored. After median sternotomy, the left ventricular
pressure (LVP) was measured using a pressure tipped Millar
catheter that was inserted through the apex into the left
ventricle. A transonic flow probe (Transonic Systems Inc.,
Ithaca, NY, USA) was placed around the proximal aorta to
measure cardiac output. Sutures were then tightened to
permanently occlude the proximal LCx. Internal defibrilla-
tion with 50 J was used when VF occurred. Prior to and 1 h
after induction of the infarct, echocardiography was
performed. After stabilization of hemodynamic parameters
and heart rhythm, the thorax was closed and the animals
were allowed to recover. Three weeks after induction of
L. Timmers et al.
myocardial infarction, the animals were anesthetized once
more and the sternum was reopened. Echocardiography and
conductance catheter-based pressure–volume (PV) loop
recordings were obtained to assess cardiac function and
geometry. After the functional measurements the heart was
excised for laboratory analysis.
Immunohistochemistry
Three weeks following MI, myocardial biopsies were obtained
from the infarct area, border area, and remote area and
fixated in 4% formalin for 24 h before being embedded in
paraffin. To determine capillary density, a lectin staining,
delineating the endothelium, was performed following
antigen retrieval by boiling in 10 mM citrate buffer.
Endogenous biotin was blocked using 0.1% avidin solution
(DakoCytomation Biotin Blocking system X0590) for 15 min at
room temperature (RT) and subsequently with 0.01% biotin
solution (Dako) for 15 min at RT. After blocking with 3.0% BSA
in PBS, the sections were incubated overnight with biotiny-
lated BS-1 (Sigma, L3759) in a 1:300 dilution in PBS with 0.1%
BSA and subsequently with streptavidin-HRPO (1:1000 in PBS)
for 1 h at RT. Finally, the sections were incubated for 30 min
in 3-amino-9-ethylcarbazole (AEC) and stained with hema-
toxylin before being embedded. Capillary density was
expressed as the number of capillaries per square millimeter
tissue. Quantification of collagen density was performed
using picrosirius red staining with circularly polarized light
and digital image microscopy after conversion into gray-
value images, as described before (Timmers et al., 2007b).
Myocardial infarct size
After excision of the heart, the LV was isolated and cut into 5
slices from apex to base. To discriminate infarct tissue from
viable myocardium, the slices were incubated in 1%
triphenyltetrazolium chloride (TTC, Sigma-Aldrich Chemi-
cals, Zwijndrecht, Netherlands) in 37 °C Sörensen buffer
(13.6 g/L KH2PO4 + 17.8 g/L Na2H PO4·2H2O, pH 7.4) for
15 min. All slices were scanned from both sides and in each
slide the infarct area was compared to total area using digital
planimetry software. After correction for the weight of the
slices, infarct size was calculated as a percentage of the LV.
Cardiac function
Midpapillary short axis epicardial echocardiography was
performed before coronary artery ligation, 1 h after and
3 weeks after coronary artery ligation (Prosound SSD-5000,
5 MHz probe UST-5280-5, Aloka Holding Europe AG, Zug,
Switzerland). Wall thickness (WT) of the infarct area and left
ventricular internal area (LVia) were measured at end-
diastole (ED) and end-systole (ES). Systolic wall thickening
(SWT) was calculated as (WT(ES) – WT(ED))/WT(ED) * 100 (%)
and fractional area shortening (FAS) as (LVia(ED) – LVia(ES))/
LVia(ED) * 100 (%). Left ventricular (LV) pressure–volume
loops were measured using a conductance catheter as
described previously (Timmers et al., 2009). Diastolic
chamber stiffness was quantified by means of linear
regression of the end-diastolic pressure–volume relationship
(Sagawa, 1981).

Data analysis
The data were collected and analyzed in a blinded fashion.
Data are presented as mean ± SE. Mortality was compared
using Fisher's exact test. Capillary sprouting was compared
using a one-way ANOVA and Bonferroni post hoc tests. A two-
way ANOVA with Bonferroni post hoc tests was used to
compare capillary density and collagen density in the
myocardial tissue. Cardiac functional parameters that were
also measured before MI (baseline) were compared using a
two-way ANOVA for repeated measures and Bonferroni post
hoc tests. For cardiac functional parameters that were
measured only 3 weeks after MI, Student's t test was used.
Acknowledgments
This work was supported by the Netherlands Heart Founda-
tion [Grant 2005T022, L.T.] and an Internationalization grant
by the University Medical Center Utrecht, the Netherlands
[D.P.V.K.].
Appendix A. Supplementary data
Supplementary data to this article can be found online at
doi:10.1016/j.scr.2011.01.001.
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