Am J Physiol Heart Circ Physiol 304: H885–H894, 2013.
First published January 11, 2013; doi:10.1152/ajpheart.00587.2012.
Placental growth factor increases regional myocardial blood flow and
contractile function in chronic myocardial ischemia
Xiaoshun Liu,1* Piet Claus,1* Ming Wu,1 Geert Reyns,2 Peter Verhamme,1 Peter Pokreisz,1
Sara Vandenwijngaert,1 Christophe Dubois,1 Johan Vanhaecke,1 Erik Verbeken,4 Jan Bogaert,3
and Stefan Janssens1
1
Division of Clinical Cardiology and Department of Cardiovascular Sciences, Gasthuisberg University Hospitals, Leuven,
Belgium; 2Vesalius Research Center, Flemish Institute of Biotechnology, University of Leuven, Leuven, Belgium; 3Department
of Radiology, Gasthuisberg University Hospitals, Leuven, Belgium; and 4Department of Pathology, Gasthuisberg University
Hospitals, Leuven, Belgium
Submitted 2 August 2012; accepted in final form 3 December 2012
chronic myocardial ischemia; growth factors; angiogenesis
CORONARY ARTERY DISEASE is the leading cause of death and
disability in the developed world (36). The prevalence and
severity of chronic myocardial ischemia in patients with coronary artery disease emphasize the limitations of current revascularization strategies. In most patients with advanced atherosclerosis (3, 37), the native angiogenic response fails to
*X. Liu and P. Claus contributed equally to this work.
Address for reprint requests and other correspondence: S. Janssens, Div. of
Clinical Cardiology, Dept. of Cardiovascular Diseases, Campus Gasthuisberg,
Univ. of Leuven, Herestraat 49, Leuven B-3000, Belgium (e-mail: Stefan.
[email protected]).
http://www.ajpheart.org
create an effective biological bypass, highlighting the need for
improved therapies for refractory ischemia and ischemic myocardial dysfunction (4, 5). Vascular growth in ischemic tissue
is a homeostatic response to maintain O2 tension and nutrient
supply and is tightly regulated by angiogenic growth factors.
Preclinical studies (15, 21, 29) have shown that administration
of single angiogenic growth factors, including FGF2 or
VEGF165, transiently improves myocardial flow and function
in porcine and canine models. However, clinical experience
with these agents has been less successful, in part attributable
to suboptimal delivery or dose-dependent side effects (16, 19,
22, 23, 33).
Placental growth factor (PlGF) has a distinct biological
profile with a predominant role in pathological angiogenesis
without affecting quiescent vessels in healthy organs (6, 25).
Genetic studies (6, 8) in mice have shown that PlGF, a member
of the VEGF family, binds to VEGF receptor (VEGFR)-1
(Flt-1) but not to VEGFR-2 (Flk1) and can accelerate murine
wound healing without increasing vascular permeability, a potentially attractive therapeutic strategy for chronic ischemia-induced
myocardial dysfunction. The aim of this study was to evaluate the
therapeutic potential of recombinant human (rh)PlGF in a porcine
model of chronic ischemic cardiomyopathy, representative of
human disease. We evaluated whether sustained systemic administration of rhPlGF (15 ␮g·kg⫺1·day⫺1) improves myocardial
neovascularization and enhances cardiac perfusion and regional
function during ischemic stress.
MATERIALS AND METHODS
Experiments in pigs were performed in accordance with Belgium
National Institute of Health guidelines for the care and use of laboratory animals, and the protocol of this double-blind randomized
controlled study was approved by the local Ethics Committee on
Animal Research (Ethische Commissie Dierproeven) of the University of Leuven (Leuven, Belgium).
Animal preparation and catheterization. Domestic cross-bred pigs
of either sex (Sus scrofa, weight: 22–25 kg) were premedicated with
aspirin (300 mg/day, Dispril, Reckitt Benckiser, Brussels, Belgium)
and clopidogrel (300 mg/day, Plavix, Sanofi, Paris, France) 1 day
before and on the day of the procedure. Pigs were sedated using
azaperone (3 mg/kg im, Stresnil, Janssen Pharmaceutics, Beerse,
Belgium) and anesthetized using ketamine (1 mg/kg iv, Eurovet,
Heusden-Zolder, Belgium) followed by a 10 mg·kg⫺1·h⫺1 continuous
infusion of propofol (Diprivan, AstraZeneca, Destelbergen, Belgium)
and mechanically ventilated using 50% O2. Continuous electrocardiographic monitoring of heart rate, rhythm, and S-T segment changes
was performed. Coronary angiograms were performed after an intra-
0363-6135/13 Copyright © 2013 the American Physiological Society
H885
Downloaded from http://ajpheart.physiology.org/ by 10.220.33.5 on June 16, 2017
Liu X, Claus P, Wu M, Reyns G, Verhamme P, Pokreisz P,
Vandenwijngaert S, Dubois C, Vanhaecke J, Verbeken E, Bogaert
J, Janssens S. Placental growth factor increases regional myocardial
blood flow and contractile function in chronic myocardial ischemia.
Am J Physiol Heart Circ Physiol 304: H885–H894, 2013. First
published January 11, 2013; doi:10.1152/ajpheart.00587.2012.—Placental growth factor (PlGF) has a distinct biological phenotype with a
predominant proangiogenic role in disease without affecting quiescent
vessels in healthy organs. We tested whether systemic administration
of recombinant human (rh)PlGF improves regional myocardial blood
flow (MBF) and systolic function recovery in a porcine chronic
myocardial ischemia model. We implanted a flow-limiting stent in the
proximal left anterior descending coronary artery and measured systemic hemodynamics, regional myocardial function using MRI, and
blood flow using colored microspheres 4 wk later. Animals were then
randomized in a blinded way to receive an infusion of rhPlGF (15
␮g·kg⫺1·day⫺1, n ⫽ 9) or PBS (control; n ⫽ 10) for 2 wk. At 8 wk,
myocardial perfusion and function were reassessed. Infusion of
rhPlGF transiently increased PlGF serum levels ⬎30-fold (1,153 ⫾
180 vs. 33 ⫾ 18 pg/ml at baseline, P ⬍ 0.001) without affecting
systemic hemodynamics. From 4 to 8 wk, rhPlGF increased regional
MBF from 0.46 ⫾ 0.11 to 0.85 ⫾ 0.16 ml·min⫺1·g⫺1, with a
concomitant increase in systolic wall thickening from 11 ⫾ 3% to
26 ⫾ 5% in the ischemic area. In control animals, no significant
changes from 4 to 8 wk were observed (MBF: 0.45 ⫾ 0.07 to 0.49 ⫾
0.08 ml·min⫺1·g⫺1 and systolic wall thickening: 14 ⫾ 4% to 18 ⫾
1%). rhPlGF-induced functional improvement was accompanied by
increased myocardial neovascularization, enhanced glycogen utilization, and reduced oxidative stress and cardiomyocyte apoptosis in the
ischemic zone. In conclusion, systemic rhPlGF infusion significantly
enhances regional blood flow and contractile function of the chronic
ischemic myocardium without adverse effects. PlGF protein infusion
may represent an attractive therapeutic strategy to increase myocardial
perfusion and energetics in chronic ischemic cardiomyopathy.
H886
PlGF INCREASES CARDIAC FLOW AND FUNCTION IN CHRONIC ISCHEMIA
ASA (300mg)
Clop (300mg)
ASA 300mg/d; Clop 75mg/d
ASA (450mg)
Heparin (10000IU)
D-1 D 0
Randomized treatment
(PBS or PlGF (15µg/kg/d)
4W
6W
8W
Evaluation of function &
Evaluation of
functional analysis
regional blood flow
treatment effect
(MRI, microspheres)
(MRI, microspheres, histology)
Fig. 1. Study design. Pigs underwent percutaneous interventions on day 0, week 4, and week 8 and MRI scans on weeks 4 and 8. Randomized treatment [PBS
vs. placental growth factor (PlGF)] started at 4 wk and was continued for 2 wk. D, day; W, week; ASA, acetylsalicylic acid; Clop, clopidogrel.
coronary administration of nitroglycerin (200 ␮g) at baseline and 4and 8-wk followups.
At baseline, after an intravenous bolus of heparin (10,000 IU,
Heparine Leo, Leo Pharma, Wilrijk, Belgium) and acetylsalicylic acid
(450 mg), a tight proximal left anterior descending coronary artery
(LAD) stenosis was induced by implantation of a flow-limiting stent.
In the central part of this homemade stent, flow limitation was
obtained by wiring a 0.017-mm stainless steel wire for 15 convolutions, which effectively prevented expansion of the central portion of
the stent upon balloon inflation (Fig. 1). At 8 wk, pigs were euthanized
using an overdose of propofol and saturated KCl. The left ventricle
(LV) distal to the stent was sectioned into five slices perpendicular to
the myocardial long axis from the base to apex for infarct staining,
microsphere analysis of myocardial blood flow, quantitative PCR, and
histology.
Assessment of hemodynamics, perfusion, and function. During
catheterization at 4 and 8 wk, a 5-Fr pressure transducer catheter
(Millar Instruments) was inserted into the LV for hemodynamic
measurements. After hemodynamic data were obtained, a 6-Fr pigtail
catheter was inserted in the LV, and 2 million colored microspheres
(15-␮m diameter, Triton Technologies) were injected to measure
regional myocardial blood flow (20). Absolute blood flow was quantified by comparing microsphere concentrations in different myocardial regions with those measured in a reference blood sample drawn
from the carotid artery using an automated pullback. The withdrawal
pump was set at a speed of 7 ml/min, started 10 s before microsphere
injection, and maintained for 3 min. Two reference tissue samples
were obtained from the right and left kidneys to test homogeneity of
the microsphere distribution. Myocardial samples from the ischemic
and remote areas (posterior wall) and reference blood and kidney
samples were analyzed using a luminescence spectrophotometer (Agilent 8453E UV-visible spectroscopy system) (20).
Cardiac MRI was performed on a 3-T system (TRIO-Tim, Siemens) at
4 and 8 wk using electrocardiographic triggering and cardiac-dedicated
surface coils. Global and regional function were assessed with breathhold cine MRI in the vertical and horizontal long and short axes,
covering the complete LV by 6-mm-thick slices. Myocardial viability
was evaluated with late contrast-enhanced (LE) MRI.
A complete description of the MRI sequences has been previously
reported (41). All MRI results were analyzed using dedicated software
by two investigators (X. Liu and P. Claus) unaware of treatment
allocation. For the calculation of myocardial infarct size, endocardial
and epicardial borders and LE regions were contoured. Myocardial
infarct size was calculated as total LE volume normalized to LV
myocardial volume. For the assessment of regional and global LV
function, endocardial and epicardial borders were traced in enddiastolic and end-systolic short-axis slices. We calculated LV enddiastolic volume, LV end-systolic volume , stroke volume, and ejection fraction as an index for global function. All volumes were
reported indexed for body surface area. Myocardial wall thickening
was measured as an index of regional function in remote and ischemic
cores after the American Heart Association segmentation model
(exclusion of the two most basal and apical slices to avoid measure-
Table 1. Primers used for quantitative PCR
Gene
pVEGF receptor-1
Forward
Reverse
pFGF2
Forward
Reverse
pVEGF
Forward
Reverse
pIGF-I
Forward
Reverse
pPDGF-B
Forward
Reverse
pVEGF receptor-2
Forward
Reverse
pVEGF-A
Forward
Reverse
pHIF-1a
Forward
Reverse
pHGF
Forward
Reverse
Sequence
5=-CCCCAAAGAAAGGCCAAGATT-3=
5=-GCAGGTCGCCTAGTTTTTCCA-3=
5=-TCTTCCTGCGCATCCACC-3=
5=-TTGCACACACTCCTTTGATAGACA-3=
5=-TACCTCCACCATGCCAAGTG-3=
5=-TGTCCACCAGGGTCTCGATT-3=
5=-CATCTCTTCTACTTGGCCCTGTG-3=
5=-ACACGAACTGAAGAGCGTCCA-3=
5=-AACAACCGCAACGTGCAGT-3=
5=-CGTCACCGTGGCCTTCTTA-3=
5=-CTTCTGTAAGATGCTCACAATTCCA-3=
5=-CGACAGAGGCCATGTCAGTGT-3=
5=-CCATGCAGATTATGCGGATCA-3=
5=-TCTCTCCTATGTGCTGGCCTTG-3=
5=-CATTGCCTGCCTCTGAAACTC-3=
5=-AGGGACTCTGGATTTGGTTCTAACTT-3=
5=-ACAAGCAATCCAGAGGTACGCT-3=
5=-TGCCGGTGTGGTGTCTGAT-3=
p, porcine; HIF, hypoxia-inducible factor; HGF, hepatocyte growth factor.
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Baseline
H887
PlGF INCREASES CARDIAC FLOW AND FUNCTION IN CHRONIC ISCHEMIA
30 Pigs
Day 0
26 Pigs
with LAD reduction stent
4 Pigs
sham (no stent)
1 Pig died
3 Pigs
excluded
4w
8w
10 Pigs
PBS minipump
9 Pigs
PlGF minipump
4 Pigs
sham (no stent)
10 Pigs
PBS minipump
MRI: n=9
9 Pigs
PlGF minipump
MRI: n=8
4 Pigs
sham (no stent)
MRI: n=4
ments errors because of the presence of the LV outflow tract and
partial volume effects) (26). At 4 wk, the segment in the LAD
perfusion territory with the most reduced function was considered as
representative for the ischemic region and followed over time.
Administration of PlGF. After the confirmation of LV dysfunction
using cine MRI, animals were randomized to a 2-wk chronic intravenous infusion of rhPlGF or PBS (control; Fig. 1) via osmotic minipumps. rhPlGF protein was produced at Eurogentec under the European Sixth Framework Program Vasoplus. Circulating hPlGF levels
were quantified using a standard PlGF immunoassay. Plates were
coated overnight (4°C) with a monoclonal antibody specifically recognizing hPlGF, blocked for 1 h at room temperature with 1% BSA,
washed, and incubated with a secondary biotinylated polyclonal goat
anti-hPlGF antibody. Bound hPlGF was detected after an incubation
with a streptavidin-horseradish peroxidase substrate. Standard dilutions of rhPlGF-1 served as positive control samples.
Chemical, histopathological, and molecular analyses. Cardiac necrosis markers and liver function tests were analyzed at stent implantation and at 4 and 8 wk followups. Cardiomyocyte necrosis, hypertrophy, metabolism, neovascularization, and capillary density were
evaluated on 5-␮m sections from paraffin-embedded biopsy specimens of the ischemic and remote zones. Periodic acid-Schiff (PAS)
staining was used to assess glycogen content. Standard endothelial
cell (CD31) and smooth muscle cell [smooth muscle actin (SM
␣-actin)] markers were used to label capillaries and small muscularized vessels. The degree of neovascularization (CD31) and fibrosis
(Sirius red) was semiquantitatively evaluated by an experienced
pathologist (E. Verbeken) blinded to treatment allocation using a
scoring system with a scale of 0 –3 (where 0 ⫽ absent, 0.5 ⫽ minimal,
1 ⫽ mild, 2 ⫽ moderate, and 3 ⫽ severe) (37). Vascular density and
glycogen content were assessed by counting SM ␣-actin-positive
vessels and PAS-positive areas in 10 randomly selected high-power
fields (HPFs) in the ischemic and remote myocardium. Cross-sectional area and perimeter and diameter of cardiac myocytes and their
nuclei from the ischemic and remote zones were measured on 10
randomly selected HPFs.
Cardiomyocyte apoptosis was evaluated using cleaved caspase-3
(Cell Signaling) and TUNEL. The apoptotic rate was expressed as the
percentage of caspase-3 and TUNEL-positive nuclei over the total
number of nuclei, which was calculated from 10 randomly selected
HPFs in the respective zones. Oxidative stress was evaluated using
8-hydroxy-2=-deoxyguanosine (8-OHdG) immunohistochemistry and
immunoblot analysis for nitrotyrosine (Sanbio). Oxidative stress was
expressed as 8-OHdG-positive nuclei over the total nuclei from 10
randomly selected HPFs per region per animal. The inflammation
response was evaluated using CD45 and MAC3 immunohistochemistry. Inflammation was expressed as CD45- and MAC3-positive
nuclei over the total number of nuclei from 10 randomly selected
HPFs per region per animal.
Myocardial protein extracts were separated by SDS-PAGE using
standard techniques. The following antibodies were used: mouse antiactin monoclonal antibody (Millipore), Akt, phosphorylated (p)Akt, glycogen synthase kinase (GSK)-3␤, pGSK-3␤, and caspase-3. In addition,
transcript levels of genes encoding Flt-1 (VEGFR-1), FGF2, VEGF,
Table 2. Heart rate and blood pressure measurements at 4 and 8 wk
Control Group
Heart rate, beats/min
Mean arterial pressure, mmHg
PlGF-Treated Group
Sham Group
4 wk
8 wk
4 wk
8 wk
4 wk
8 wk
113 ⫾ 6*
97 ⫾ 6
103 ⫾ 9*
88 ⫾ 4
118 ⫾ 6*
94 ⫾ 3
109 ⫾ 3*
90 ⫾ 3
91 ⫾ 7
89 ⫾ 15
90 ⫾ 6
87 ⫾ 7
Values are means ⫾ SE; n ⫽ 10 animals in the control group, 9 animals in the placental growth factor (PlGF)-treated group, and 4 animals in the sham-operated
(sham) group. *P ⬍ 0.05 vs. the sham group.
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Fig. 2. Study flow chart. Of 30 pigs, 19 pigs
were randomized to the control group (PBS
treatment; n ⫽ 10) or PlGF-treated group
(n ⫽ 11) and 4 untreated sham-operated
(sham) pigs were included to account for
age-related changes in left ventricular (LV)
structure and function. Serial MRI was obtained in 21 animals (n ⫽ 9 animals in the
control group, 8 in the PlGF-treated group,
and 4 in the sham group).
3 Pigs died
week 1 (ischemia)
H888
PlGF INCREASES CARDIAC FLOW AND FUNCTION IN CHRONIC ISCHEMIA
Table 3. Serial regional myocardial blood flow analysis using colored microspheres
Control Group
Ischemic myocardium
Remote myocardium
PlGF-Treated Group
Sham Group (Corresponding Region)
4 wk
8 wk
4 wk
8 wk
4 wk
8 wk
0.45 ⫾ 0.07‡
0.84 ⫾ 0.07
0.49 ⫾ 0.08
1.07 ⫾ 0.09
0.46 ⫾ 0.11‡
0.73 ⫾ 0.09
0.85 ⫾ 0.16*†
1.17 ⫾ 0.11*
0.90 ⫾ 0.14
1.05 ⫾ 0.12
0.71 ⫾ 0.05
1.06 ⫾ 0.14
Values are means ⫾ SE (in ml 䡠 min⫺1 䡠 g⫺1); n ⫽ 10 animals in the control group, 8 animals in the PlGF-treated group, and 4 animals in the sham group.
*P ⬍ 0.05 vs. 4 wk; †P ⬍ 0.05 vs. the control group; ‡P ⬍ 0.05 vs. the sham group.
A
Ischemic segment
60
Wall thickening (%)
Pharmacokinetic and safety experiments after intravenous
PlGF infusion. Serum PlGF levels were measured in two
healthy pigs on day 0 and on days 5, 10, and 14 after minipump
implantation. Circulating PlGF levels were significantly elevated on day 5 (1,106 pg/ml) compared with baseline (11
pg/ml) and remained elevated on days 10 and 14 (654 and 665
pg/ml, respectively).
During chronic ischemia, serum PlGF levels at 4 wk (before
minipump implantation) and 8 wk were comparable between
control and PlGF treatment (45 ⫾ 7 vs. 84 ⫾ 49 and 52 ⫾ 18
vs. 46 ⫾ 18 pg/ml, respectively). However, PlGF-treated pigs
had ⬎30-fold higher circulating PlGF levels than control pigs
4 – 6 days after minipump implantation (33 ⫾ 18 vs. 1,153 ⫾
180 pg/ml, P ⬍ 0.001). Cardiac necrosis markers and liver
function tests remained unchanged in both groups.
Study followup. Thirty pigs were enrolled in the study
followup (4 sham-operated pigs and 26 stented pigs; Fig. 2). Of
the 26 stented pigs, 3 pigs died of acute ischemic complications, 1 pig died during baseline MRI, and 3 pigs were
excluded after 4 wk because of an absence of coronary stenosis
(n ⫽ 1) or total vessel occlusion (n ⫽ 2). In 19 pigs blindly
randomized to PlGF or PBS treatment, no adverse events were
observed during or after treatment.
PlGF improves regional myocardial blood flow and systolic
function. Compared with sham-operated animals, heart rate
was elevated in all stented animals at 4 and 8 wk, whereas
mean arterial pressure did not differ (Table 2).
At 4 wk, myocardial blood flow was reduced by half in the
ischemic territory in both groups and increased thereafter
significantly after PlGF infusion, but not in the control group
(Table 3). Reference blood flow to the kidneys was similar in
all groups at 4 and 8 wk.
Stent implantation induced very small infarction in five of
nine scanned animals in the control group (median: 6%, range:
5– 6%) and in four of eight scanned animals in the PlGF-treated
group (median: 7%, range: 2–11%). The size of myocardial
necrosis was similar in both groups and remained unchanged at
8 wk (control group: median 6% and range 5– 6% vs. PlGFtreated group: median 5% and range 2–11%).
4w
8w
50
40
*†+
30
20
+
+
+
10
0
Con (n=9)
B
Wall thickening (%)
RESULTS
Stent implantation reduced systolic wall thickening (SWT)
in the ischemic territory to the same extent in both groups at 4
wk. Subsequent PlGF transfer significantly and selectively
increased SWT in the ischemic areas at 8 wk, whereas PBS
treatment had no effect (Fig. 3). In sham-operated animals,
there were no significant changes in SWT over time in the
LAD perfusion territory (55 ⫾ 1% at 4 wk vs. 53 ⫾ 1% at 8
wk) or in segments corresponding to remote areas in stented
pigs (57 ⫾ 1% at 4 wk vs. 54 ⫾ 2% at 8 wk). The reduction
in SWT in stented pigs at 4 wk was associated with an
increased LV end-systolic volume index and reduced stroke
volume index. At 8 wk, there were no significant differences in
global LV function (Table 4).
PlGF induces neovascularization in the ischemic myocardium.
Clusters of SM ␣-actin-positive vessels were observed in the
ischemic myocardium (Fig. 4, A and B) but not in the remote
myocardium. Quantitative analysis showed a significantly
higher number and larger area of SM ␣-actin positive vessels
on randomly selected HPFs in the PlGF-treated group (Fig. 4,
A–D). Capillary density score, measured from the number of
lectin-positive clusters, was greater in ischemic areas from the
60
PlGF (n=8)
Sham (n=4)
Remote segment
4w
8w
50
40
+
+
+
+
30
20
10
0
Con (n=9)
PlGF (n=8)
Sham (n=4)
Fig. 3. PlGF treatment significantly improves regional LV systolic wall
thickening. Changes in regional wall thickening are shown between 4 and 8 wk
in the ischemic (A) and remote (B) segments and in matched segments of sham
animals. *P ⬍ 0.05 vs. the control (Con) group; †P ⬍ 0.05 vs. 4 wk; ⫹P ⬍
0.05 vs. the sham group.
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IGF-I, PDGF-B, VEGFR-2, VEGF-A, hypoxia-inducible factor-1a, and
hepatocyte growth factor were analyzed using quantitative PCR (Table 1).
Statistical analysis. Statistical analysis was performed using SAS
statistical software (SAS version 9.2, SAS Institute). Data are expressed as means ⫾ SE. ANOVA and a Bonferroni’s posthoc test
were used to analyze differences between groups. Repeated-measures
ANOVA was used to test serial data. When data did not follow a
normal distribution, Kruskal-Wallis nonparametric statistics were reported, and differences between groups were identified using MannWhitney or Wilcoxon tests.
H889
PlGF INCREASES CARDIAC FLOW AND FUNCTION IN CHRONIC ISCHEMIA
Table 4. MRI analysis of global LV function
Control Group
Body weight, kg
LV mass index, g/m2
End-diastolic volume index, ml/m2
End-systolic volume index, ml/m2
Stroke volume index, ml/m2
Ejection fraction, %
PlGF-Treated Group
Sham Group
4 wk
8 wk
4 wk
8 wk
4 wk
8 wk
45 ⫾ 2
77 ⫾ 4
82 ⫾ 9
52 ⫾ 7†
30 ⫾ 2†
38 ⫾ 2†
65 ⫾ 2*
81 ⫾ 3
76 ⫾ 6
45 ⫾ 4†
31 ⫾ 2†
41 ⫾ 2†
45 ⫾ 1
80 ⫾ 3
81 ⫾ 5
51 ⫾ 4†
30 ⫾ 2†
37 ⫾ 3†
63 ⫾ 3*
80 ⫾ 7
81 ⫾ 9
49 ⫾ 7†
32 ⫾ 2†
41 ⫾ 3†
43 ⫾ 4
74 ⫾ 5
83 ⫾ 4
38 ⫾ 2
44 ⫾ 4
54 ⫾ 3
64 ⫾ 7*
74 ⫾ 6
80 ⫾ 8
35 ⫾ 2
44 ⫾ 7
54 ⫾ 4
Values are means ⫾ SE; n ⫽ 9 animals in the control group, 8 animals in the PlGF-treated group, and 4 animals in the sham group. Volumetric parameters
were indexed to body surface area (BSA) using Meeh’s formula as follows: BSA (in m2) ⫽ k ⫻ [body weight (in kg)]2/3 ⫻ 10⫺2, where k ⫽ 9.0 (see Table
7.1 in Ref. 31). *P ⬍ 0.05 vs. 4 wk; †P ⬍ 0.05 vs. the sham group.
B
A
PlGF preserves nutrient myocardial perfusion and is associated
with reduced cardiomyocyte cell apoptosis. To investigate the effect
of PlGF on myocardial nutrient blood flow, we compared glycogen
deposition in ischemic and remote areas. The glycogen area (%HPF)
in the ischemic zone of control was greater than the glycogen area in
the PlGF-treated group (2.1 ⫾ 0.74 vs. 0.81 ⫾ 0.22, P ⬍ 0.05; Fig.
6, A–C). No differences in fibrosis were observed.
Immunohistochemical staining for 8-OHdG, an index of
ischemia-induced oxidative DNA damage, showed a significantly greater percentage of 8-OHdG-positive nuclei in the
ischemic area of the control group than in the PlGF-treated
group (Fig. 6, D–F). To investigate possible effects of PlGF on
the inflammatory/immune response, immunohistochemical
E
F
*†
50 µm
Con (n=9)
PlGF (n=9)
60
40
20
0
H
*†
Ischemic
I
Remote
J
G
80
Con (n=9)
*†
PlGF (n=9)
60
40
20
Capillary Score
Vascular density
(n SMa+/HPF)
80
SMa+ area (%HPF)
D
C
1.5
*†
Con (n=10)
PlGF (n=9)
1
0.5
0
0
Ischemic
Remote
K
Ischemic
Remote
L
Cardiomyocyte perimeter (μm)
Nuclei perimeter (μm)
Fig. 4. PlGF treatment induces neovascularization in the ischemic myocardium. Representative sections of smooth muscle ␣-actin-stained [SMa; control group
(A) and PlGF-treated group (B)] and lectin-stained [control group (E) and PlGF-treated group (F)] myocardial tissue in the ischemic area are shown. The number
and area of SMa-positive vessels (C and D) and number of lectin-positive vessels (G) were significantly higher in the ischemic area of PlGF-treated animals than
in control animals. On Sirius red-stained sections [control group (H), PlGF-treated group (I), and sham group (J)], cardiomyocyte size and nuclei perimeters were
larger in the ischemic area of the control group than in the PlGF-treated group, with a clear concomitant rightward shift in the distribution of cardiomyocyte
perimeter (K) and nuclear perimeter (L). The dashed red lines indicate mean perimeters in the sham group. HPF, high-powered field. *P ⬍ 0.05 vs. the control
group; †P ⬍ 0.05 vs. the remote myocardium.
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PlGF-treated group compared with the control group (0.64 ⫾
0.3 vs. 0.15 ⫾ 0.1, P ⬍ 0.05; Fig. 4, E–G).
Cardiomyocyte and nuclear perimeters were larger in the
control group than in the PlGF-treated group (91 ⫾ 1 vs. 79 ⫾
0.7 ␮m and 19.7 ⫾ 0.2 vs. 18.4 ⫾ 0.1 ␮m, respectively, P ⬍
0.05 for both; Fig. 4, H–L), with a marked rightward shift in
distribution (Fig. 4, K and L). There were no differences in
cardiomyocyte or nuclear sizes, cross-sectional areas, or diameters between PlGF and sham-operated group. We also measured transcript levels of several angiogenic growth factors and
their receptors in the ischemic and remote myocardium, several
of which showed increased expression levels in both the
ischemic and remote zones of PlGF-treated animals (Fig. 5).
H890
PlGF INCREASES CARDIAC FLOW AND FUNCTION IN CHRONIC ISCHEMIA
6
†
Sham (n=4)
Con(n=10)
PlGF(n=9)
†
5
4
3
*
*
†
*
pVEGFR1
staining for CD45 (Fig. 6, G–I) and MAC3 (Fig. 6, J–L) was
performed in tissue from the ischemic region. Semiquantitative
analysis showed no increases in CD45- or MAC3-positive
nuclei in PlGF-treated animals.
pVEGF
pIGF1
pPDGFb
pVEGFA
pVEGFR2
pHIF1a
Ischemic
Remote
Ischemic
Remote
Ischemic
Remote
Ischemic
Remote
†
Ischemic
Ischemic
Remote
Ischemic
Remote
Ischemic
Remote
pFGF2
*
†
pHGF
To explore molecular mechanisms of PlGF-mediated cardioprotection, we measured Akt and GSK-3␤ protein levels
and phosphorylation states in the ischemic region (Fig. 7). In
the PlGF-treated group, a twofold higher ratio of pAkt to Akt
A
D
G
J
B
E
H
K
50 µm
8-OHdG positive/ total
nuclei (%)
Glycogen area
(%HPF)
2.5
2
1.5
1
*
0.5
0
Ischemic
F
Remote
40
Con(n=5)
†
I
PlGF(n=5)
*†
20
Con (n=5)
6
5
4
3
2
1
0
0
Ischemic
Remote
L
PlGF (n=4)
MAC3 positive/ total
nuclei (%)
Con (n=10)
PlGF (n=9)
3
CD45 positive/ total
nuclei (%)
C
Ischemic
Remote
4
3.5
3
2.5
2
1.5
1
0.5
0
Con (n=5)
PlGF(n=4)
Ischemic Remote
Fig. 6. PlGF treatment is associated with reduced glycogen deposition and cellular injury in the ischemic myocardium. Glycogen content, measured on periodic
acid-Schiff-stained myocardial tissue in the ischemic border [control group (A) and PlGF-treated group (B)], was significantly lower in PlGF-treated animals (C).
Oxidative stress was measured on 8-hydroxy-2=-deoxyguanosine (8-OHdG)-stained sections from ischemic tissue [control group (D) and PlGF-treated group (E)].
The percentage of 8-OHdG-positive nuclei was significantly lower in PlGF-treated animals (F). Inflammation was measured using CD45 [control group (G) and
PlGF-treated group (H)] and MAC3 [control group (J) and PlGF-treated group (K)]. No significant differences in inflammation response in the ischemic region
were observed (I and L). *P ⬍ 0.05 vs. the control group; †P ⬍ 0.05 vs. the remote myocardium.
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00587.2012 • www.ajpheart.org
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0
Ischemic
1
Remote
2
Remote
Fig. 5. PlGF treatment increases expression levels of angiogenic growth factors and receptors in
myocardial tissue of hearts subjected to chronic
ischemia. Expression levels of angiogenic growth
factors and receptors in the ischemic and remote
myocardium of sham, control, and PlGF-treated
pigs are shown. p, porcine; VEGFR, VEGF receptor; HIF, hypoxia-inducible factor; HGF, hepatocyte growth factor; Ct, threshold cycle. *P ⬍
0.05 vs. the control group; †P ⬍ 0.05 vs. the
sham group.
Expression (2-∆∆ ∆ Ct)
†: p<0.05 vs Sham; *: p<0.05 vs Con
PlGF INCREASES CARDIAC FLOW AND FUNCTION IN CHRONIC ISCHEMIA
and pGSK-3␤ to GSK-3␤ was observed than in corresponding
ischemic regions of the control group, whereas in remote
regions or in healthy animals no differences in phosporylation
status were detected with PlGF treatment (Fig. 7). Finally,
PlGF was associated with reduced apoptosis, as evidenced by
twofold lower caspase 3 expression (Fig. 8, A and B) and a
significantly lower percentage of TUNEL- and caspase-3positive nuclei in the ischemic area compared with the control
group (Fig. 8, C–H).
DISCUSSION
38) and variable improvements in LV function (21, 32). Subsequent studies (13, 16, 19, 22, 23, 32, 33) using FGF or VEGF
in patients with ischemic heart disease failed to recapitulate
preclinical findings, in part because of ineffective delivery
strategies, insufficient duration of biological activity after
short-term intracoronary administration of recombinant proteins, or dose-related side effects. The latter were attributed to
systemic exposure to potent growth factors, resulting in hypotension, glomerulotoxicity, and anemia due to bone marrow
suppression (13, 16, 32).
Here, we tested the potential of sustained systemic delivery
of PlGF to stimulate perfusion and improve regional contractile
function in the chronic ischemic myocardium. PlGF acts via
VEGFR-1 (Flt-1) binding on endothelial cells, smooth muscle
cells, macrophages, and bone marrow progenitor cells and has
a unique role in vascular development under pathological
conditions. In rodent models of limb ischemia, sustained infusion of PlGF for 3–7 days accelerated collateral growth in
ischemic limbs without prohibitive side effects (5, 6, 28) and
enhanced functional recovery (25). Similarly, after permanent
coronary artery ligation in mice, a single high-dose intramuscular injection induced angiogenesis in the border zone of the
infarct (35). While these permanent ligation models are less
representative of human disease, our porcine coronary stenosis
model recapitulates some of the structural and functional characteristics of ischemic cardiomyopathy in patients (10) and
integrates MRI-based functional assessments, which are routinely used in clinical practice.
PlGF enhances neovascularization and perfusion in the
ischemic myocardium. PlGF administration improved regional
blood flow to the ischemic myocardium. We confirmed that the
A
PlGF
Con
PlGF (n=4)
Con (n=4)
PlGF in
normal pigs
(n=2)
Akt
pAkt
GSK-3β
pGSK-3β
Actin
Ischemic region
Non-ischemic (remote) region
Ischemic
2.4
C
Non-ischemic
pGSK-3β/ GSK-3β
B
pAkt/Akt
*
1.6
0.8
0
2.4
Ischemic
Non-ischemic
1.6
Fig. 7. Immunoblot analysis of Akt and glycogen
synthase kinase (GSK)-3␤ (A) in total protein
extracts from ischemic and remote tissue and
in two normal animals 14 days after PlGF infusion in the pharmacokinetic analysis. In ischemic segments, densitometry revealed a significantly increased pAkt-to-Akt ratio (B) in PlGFtreated animals compared with control animals,
whereas in nonischemic tissue, the phophorylation status was comparable. In control animals,
the pGSK-3␤-to-GSK-3␤ ratio was reduced in
ischemic tissue (C) compared with nonischemic
regions or with the healthy myocardium. In contrast, pGSK-3␤-to-GSK-3␤ ratios did not decrease in ischemic tissue of PlGF-treated animals. *P ⬍ 0.05 vs. the control group.
*
0.8
0
Con
PlGF
Normal
PlGF
Con
PlGF
Normal
PlGF
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00587.2012 • www.ajpheart.org
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This study shows that systemic administration of rhPlGF
enhances myocardial perfusion, regional LV function, and
energy metabolism in the chronic ischemic myocardium. Resting blood flow to the anterior wall was reduced by half, and
subsequent ischemia-induced LV dysfunction was documented
with MRI in a preclinical porcine model. Chronic infusion of
rhPlGF was well tolerated and enhanced myocardial blood
flow and neovascularization in the ischemic territory. Importantly, enhanced perfusion after PlGF administration resulted
in improved systolic function recovery in the ischemic area,
which was associated with reduced cardiomyocyte apoptosis.
Growth factor-mediated therapies for ischemic cardiomyopathy.
Therapeutic angiogenesis for ischemic cardiovascular disease
is based on the concept that stimulated coronary collateral
development from existing microvessels can alleviate myocardial ischemia and enhance functional recovery. Animal models
of myocardial infarction (15) or ameroid constrictor-induced
chronic myocardial ischemia (2, 14, 15, 21, 29, 38) have shown
increased collateral blood flow after administration of members
of the FGF and VEGF growth factor families (2, 14, 15, 21, 29,
H891
H892
A
P l GF
B
C on
Caspase-3
Actin
Relative density
PlGF INCREASES CARDIAC FLOW AND FUNCTION IN CHRONIC ISCHEMIA
1.5
*
1
0.5
0
Con (n=5)
40
*
30
Non-ischemic
20
10
0
Con (n=5)
H
20 µm
Apoptosis index
(TUNEL)
G
TUNEL
F
Ischemic
PlGF (n=5)
Ischemic
Non-ischemic
40
*
30
20
10
0
Con (n=5)
PlGF (n=5)
Fig. 8. PlGF treatment is associated with reduced apoptosis in the ischemic myocardium. Caspase-3 immunoreactivity in the ischemic myocardium was 2.4-fold
higher in extracts from the control group (A and B). Also, immunohistochemical staining using caspase-3-specific antibody (C and D) and TUNEL (F and G)
revealed a greater apoptotic index in the ischemic zone of control animals (E and H). *P ⬍ 0.05 vs. PlGF.
chronic ischemic burden per se did not trigger a major endogenous PlGF release at 4 wk, which could confound the observed neovascularization. Endogenous PlGF release typically
and transiently accompanies acute ischemia and infarction
(18), but reported levels are 30-fold less than levels measured
here. High circulating PlGF levels induced characteristic morphological features of angiogenesis and arteriogenesis in the
ischemic porcine myocardium (Fig. 4). Potential mechanisms
include the muscularization of small neovessels via the
release of smooth muscle cell mitogens from activated
endothelial cells and fibroblasts (1), from infiltrating inflammatory cells (7), or via direct Flt-1 receptor binding on
smooth muscle cells (24) and stimulated growth of arterial
collaterals (39). In this study, we did not detect a difference
in infiltrating monocytes or macrophages in the ischemic
tissue 4 wk after PlGF infusion.
Interestingly, we detected increased expression levels of
several growth factors and receptors in PlGF-treated animals
(Fig. 5), the mechanism of which is unknown. Preclinical
studies have shown that some of these growth factors, such as
hepatocyte growth factor, in turn increase the expression of
VEGFRs and c-Met in endothelial cells (30, 40) and cooperate
with VEGF-A165 to amplify the vasculoproliferative response
(12, 34). To what extent such growth factor cooperativity
mediates the effects of PlGF in the ischemic myocardium
remains to be determined. We also observed higher pAkt and
pGSK-3␤ protein levels in PlGF-treated animals (Fig. 7, B and
C), and several groups (17, 27) have previously reported that
GSK-3␤, when phosphorylated by Akt at Ser9, is strongly
cardioprotective and associated with reduced apoptosis.
Study limitations. We studied juvenile pigs, precluding direct extrapolation to patients with advanced age, comorbidities,
and atherosclerosis, in whom angiogenic growth factors could
induce intimal hyperplasia and progression of coronary atherosclerotic lesions (9). We used a single sustained delivery of
rhPlGF and cannot exclude more effective doses or duration of
delivery.
Similarly, we only measured regional function 4 wk after
PlGF infusion. Hence, the question whether a longer followup
would further normalize SWT and translate into improved
global function recovery remains to be addressed in future
studies.
Conclusions. In summary, prolonged systemic administration of rhPlGF in a porcine model of chronic ischemic cardiomyopathy significantly improves regional myocardial blood
flow and contractile function without major adverse effects.
PlGF appeared to be well tolerated, and its neovascularization
capacity and metabolic effects represent an attractive therapeutic strategy in patients with chronic ischemic cardiomyopathy
and refractory myocardial ischemia.
ACKNOWLEDGMENTS
The authors thank Ellen Caluwe, Stefan Ghysels, Hilde Gillijns, and Nina
Vanden Driessche for expert technical assistance.
GRANTS
This work was supported by Flemish Government Grants IWT/040312/
O&OPlGF-THROMB-xfZL360101, University of Leuven Grant PF10/014,
and European Sixth Framework Programme Grant LSHM-CT-2006-037254
(to X. Liu and S. Janssens). S. Janssens is a Clinical Investigator of the Fund
AJP-Heart Circ Physiol • doi:10.1152/ajpheart.00587.2012 • www.ajpheart.org
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20 µm
Apoptosis index
(caspase-3)
E
D
Caspase-3
C
PlGF (n=5)
PlGF INCREASES CARDIAC FLOW AND FUNCTION IN CHRONIC ISCHEMIA
for Scientific Research-Flanders and holder of a chair supported by AstraZeneca.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: X.L. and G.R. performed experiments; X.L., P.C.,
M.W., P.P., S.V., E.V., and S.P.J. analyzed data; X.L. prepared figures; X.L.
and P.C. drafted manuscript; X.L., P.C., M.W., G.R., P.V., P.P., S.V., C.D.,
J.V., E.V., J.B., and S.P.J. approved final version of manuscript; P.C., P.P.,
J.V., and S.P.J. conception and design of research; P.C., P.V., P.P., C.D., J.V.,
E.V., J.B., and S.P.J. interpreted results of experiments; P.C., M.W., G.R.,
P.V., P.P., S.V., C.D., J.V., E.V., J.B., and S.P.J. edited and revised manuscript.
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