Vascular Endothelial Growth Factor-B Acts as a Coronary
Growth Factor in Transgenic Rats Without Inducing
Angiogenesis, Vascular Leak, or Inflammation
Maija Bry, BMed; Riikka Kivela¨, PhD*; Tanja Holopainen, MD*; Andrey Anisimov, PhD;
Tuomas Tammela, MD, PhD; Jarkko Soronen, MSc; Johanna Silvola, MSc; Antti Saraste, MD, PhD;
Michael Jeltsch, PhD; Petra Korpisalo, MD; Peter Carmeliet, MD, PhD; Karl B. Lemstro¨m, MD, PhD;
Masabumi Shibuya, MD, PhD; Seppo Yla¨-Herttuala, MD, PhD; Leena Alhonen, PhD;
Eero Mervaala, MD, PhD; Leif C. Andersson, MD, PhD; Juhani Knuuti, MD, PhD; Kari Alitalo, MD, PhD
Background—Vascular endothelial growth factor-B (VEGF-B) binds to VEGF receptor-1 and neuropilin-1 and is
abundantly expressed in the heart, skeletal muscle, and brown fat. The biological function of VEGF-B is
incompletely understood.
Methods and Results—Unlike placenta growth factor, which binds to the same receptors, adeno-associated viral delivery
of VEGF-B to mouse skeletal or heart muscle induced very little angiogenesis, vascular permeability, or inflammation.
As previously reported for the VEGF-B167 isoform, transgenic mice and rats expressing both isoforms of VEGF-B in
the myocardium developed cardiac hypertrophy yet maintained systolic function. Deletion of the VEGF receptor-1
tyrosine kinase domain or the arterial endothelial Bmx tyrosine kinase inhibited hypertrophy, whereas loss of VEGF-B
interaction with neuropilin-1 had no effect. Surprisingly, in rats, the heart-specific VEGF-B transgene induced
impressive growth of the epicardial coronary vessels and their branches, with large arteries also seen deep inside the
subendocardial myocardium. However, VEGF-B, unlike other VEGF family members, did not induce significant
capillary angiogenesis, increased permeability, or inflammatory cell recruitment.
Conclusions—VEGF-B appears to be a coronary growth factor in rats but not in mice. The signals for the
VEGF-B–induced cardiac hypertrophy are mediated at least in part via the endothelium. Because cardiomyocyte damage
in myocardial ischemia begins in the subendocardial myocardium, the VEGF-B–induced increased arterial supply to this
area could have therapeutic potential in ischemic heart disease. (Circulation. 2010;122:1725-1733.)
Key Words: angiogenesis


coronary disease


hypertrophy
Coronary artery disease leads to compromised myocardialblood supply and the typical symptoms of stress-induced
angina. Although pharmaceutical therapy and revasculariza-
tion of stenotic epicardial coronary arteries are the standard
therapy for coronary artery disease, many patients with
advanced disease or small-vessel disease respond poorly to
these treatments. Novel therapeutic strategies for promoting
collateral artery formation, or arteriogenesis, are in high
demand.1,2 Although vascular endothelial growth factor
(VEGF) is the most potent angiogenic factor for possible
therapy of myocardial ischemia,3 it also promotes vascular
leakage, inflammation, and the formation of angioma-like
vascular structures,4–7 which has hampered its utility in
therapeutic angiogenesis. Among the VEGF family members,
placenta growth factor (PlGF) has shown the most promise
for promoting therapeutic arteriogenesis in preclinical
studies.7
Clinical Perspective on p 1733
VEGF-B, isolated in 1995,8 has been an exceptional
member of the VEGF family in that efforts to discover a
blood vascular function for VEGF-B have had largely nega-
Received March 29, 2010; accepted August 18, 2010.
From the Molecular/Cancer Biology Laboratory and Institute for Molecular Medicine Finland (M.B., R.K., T.H., A.A., T.T., J. Soronen, M.J., K.A.)
and Department of Pathology (L.C.A.), Haartman Institute, Biomedicum Helsinki, University of Helsinki and Helsinki University Central Hospital,
Helsinki, Finland; Turku PET Centre, Turku University Hospital, Turku, Finland (J. Silvola, A.S., J.K.); Institute of Biomedicine, Pharmacology,
University of Helsinki, Helsinki, Finland (E.M.); Biotechnology and Molecular Medicine, A.I. Virtanen Institute for Molecular Sciences, Biocenter
Kuopio, University of Eastern Finland, Kuopio, Finland (P.K., S.Y.-H., L.A.); Department of Molecular Oncology, Tokyo Medical and Dental University,
Tokyo, Japan (M.S.); Cardiopulmonary Research Group, Transplantation Laboratory, University of Helsinki and Department of Cardiothoracic Surgery,
Helsinki University Central Hospital, Helsinki, Finland (K.B.L.); and Vesalius Research Center, KU Leuven, Leuven, Belgium (P.C.).
*Drs Kivela¨ and Holopainen contributed equally to this article.
The online-only Data Supplement is available with this article at http://circ.ahajournals.org/cgi/content/full/CIRCULATIONAHA.110.957332/DC1.
Correspondence to Kari Alitalo, Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, PO Box 63 (Haartmaninkatu 8), FI-00014 Helsinki,
Finland. E-mail kari.alitalo@helsinki.fi
© 2010 American Heart Association, Inc.
Circulation is available at http://circ.ahajournals.org DOI: 10.1161/CIRCULATIONAHA.110.957332
1725

tive results.6 VEGF-B knockout (KO) mice are viable and
display at most mild cardiac phenotypes such as a slightly
smaller heart size in 1 genetic background or a mild delay in
atrioventricular coupling in another strain.9,10 Transgenic
(TG) or adenoviral overexpression of VEGF-B in the skin or
skeletal muscle increased blood vessel density only minimal-
ly.11,12 Interestingly, however, strong overexpression of
VEGF-B by adenoviral transduction induced heart-specific
capillary dilation and increased collateral blood vessel growth
after myocardial infarction in pigs.13
VEGF-B is expressed as 2 RNA splice isoforms.14 Recently,
we demonstrated that when overexpressed in the mouse heart,
the heparin-binding VEGF-B167 isoform induced cardiac hyper-
trophy without overt angiogenesis, although it caused mild
enlargement of myocardial blood capillaries.12 The other iso-
form, VEGF-B186, is O-glycosylated, proteolytically processed,
and freely diffusible. Both isoforms bind to and activate VEGF
receptor-1 (VEGFR-1) and neuropilin-1.15,16 Several studies
have indicated that the VEGF-B isoforms are expressed simul-
taneously in various tissues.14 PlGF binds to the same receptors
as VEGF-B and has been shown to promote angiogenesis and
arteriogenesis in pathological conditions.17
The aim of this study was to investigate the mechanisms
of VEGF-B action in the heart in more depth. A critical
question about the effects of VEGF-B in the myocardium
is how they differ from those of PlGF and whether
VEGF-B could provide a means to improve myocardial
function in the failing heart. Because adenoviral transduc-
tion in an immunocompetent host is short-lived and results
in nonspecific inflammation, we have instead used adeno-
associated viral and TG delivery of VEGF-B to the mouse
and rat heart.
Methods
A detailed description of the methods used is provided in the
online-only Data Supplement.
Construction and Preparation of the Recombinant
Adeno-Associated Virus Vectors
Mouse VEGF120, PlGF, VEGF-B167, VEGF-B186, and human serum
albumin complementary DNAs were cloned into the psubCMV-
WPRE recombinant adeno-associated virus (AAV) expression vec-
tor. AAV particles were injected into tibialis anterior muscles or the
left ventricle.
Generation of
MHC–VEGF-B TG Mice and Rats
A fragment of the human VEGF-B gene and the mouse VEGF-
BEx1–5 fragment were isolated and cloned into the
-myosin heavy
chain (
MHC) promoter expression vector (a kind gift from Dr
Jeffrey Robbins). TG animals were generated by microinjection of
fertilized oocytes from FVB/N mice or HsdBrl:WH Wistar rats. All
animal experiments were approved by the Provincial State Office of
Southern Finland and carried out in accordance with institutional
guidelines.
Immunohistochemistry, Microscopy, and
Image Analysis
The antibodies and methods used are detailed in the online-only Data
Supplement.
Blood Pressure Measurements
and Echocardiography
Blood pressure was measured with the CODA Non-Invasive Blood
Pressure System for Mice and Rats (Kent Scientific Corp, Tor-
rington, Conn). Transthoracic echocardiography was performed with
an Acuson Sequoia 512 Ultrasound System and an Acuson Linear
15L8 transducer (Siemens Medical Solutions, Mountain View,
Calif).
Micro–Computed Tomography Imaging of the
Cardiac Vessels
Coronary angiographies were performed with the Inveon micro–
computed tomography scanner (Siemens, Knoxville, Tenn). The
ascending aorta was cannulated and clamped, and iodinated intra-
vascular contrast agent eXIATM160XL (Binitio Biomedical Inc,
Ottawa, Ontario, Canada) was carefully injected manually to fill the
cardiac blood vessels, avoiding very high pressure.
Assessment of Myocardial Perfusion, Oxygen
Consumption, and Efficiency of Work
Eight rats were given a slow bolus of 30
24 MBq of [11C]acetate
and imaged with the Inveon micro–positron emission tomography
scanner (Siemens, Knoxville, Tenn).
Statistical Analysis
Values are presented as mean
SD unless otherwise indicated.
Statistical analysis was performed with 1-way ANOVA (posthoc
with Tukey test) or with the 2-tailed unpaired Student t test unless
otherwise specified in the Results. Differences were considered
statistically significant at P0.05.
Results
Unlike PlGF, VEGF-B Fails to Induce Capillary
Angiogenesis or Arteriogenesis in Mouse Skeletal
or Cardiac Muscle
To compare the effects of VEGF-B and PlGF, which bind to
VEGFR-1 and neuropilin-1, we injected recombinant AAVs
encoding VEGF-B167, VEGF-B186, and PlGF, as well as
VEGF and human serum albumin as positive and negative
controls, respectively, into mouse tibialis anterior muscles.
Immunofluorescence staining of the muscles for platelet
endothelial cell adhesion molecule (PECAM)-1 and

-smooth muscle actin (SMA) 4 weeks after injection indi-
cated strong capillary angiogenesis with primarily smooth
muscle cell– coated vessels in PlGF-injected muscles,
whereas no evidence of angiogenesis was seen in muscles
overexpressing either VEGF-B isoform (Figure 1). In the
VEGF-injected muscles, endothelial cell proliferation resem-
bled angioma formation (Figure 1). As expected on the basis
of prior work with adenoviral vectors, PlGF and VEGF
increased blood perfusion in the muscles, whereas even a
combination of VEGF-B167 and VEGF-B186 had no effect on
perfusion (Figure IA in the online-only Data Supplement).
Interestingly, Evans Blue dye injection experiments indicated
that although PlGF and VEGF increased vascular permeabil-
ity, VEGF-B167 and VEGF-B186 did not (Figure IB in the
online-only Data Supplement). Similar effects were obtained
when the same AAV vectors were expressed in the myocar-
dium (Figure II in the online-only Data Supplement). Impor-
tantly, mice injected with PlGF or VEGF vectors had to be
1726 Circulation October 26, 2010