Physiol Rev 94: 779 –794, 2014
doi:10.1152/physrev.00028.2013
VASCULAR ENDOTHELIAL GROWTH FACTOR-B IN
PHYSIOLOGY AND DISEASE
Maija Bry, Riikka Kivelä, Veli-Matti Leppänen, and Kari Alitalo
Wihuri Research Institute and Translational Cancer Biology Program, University of Helsinki, Helsinki, Finland
Bry M, Kivelä R, Leppänen V-M, Alitalo K. Vascular Endothelial Growth Factor-B in Physiology and Disease. Physiol Rev 94: 779–794, 2014; doi:10.1152/physrev.00028.2013.—
Vascular endothelial growth factor-B (VEGF-B), discovered over 15 years ago, has long
been seen as one of the more ambiguous members of the VEGF family. VEGF-B is
produced as two isoforms: one that binds strongly to heparan sulfate in the pericellular
matrix and a soluble form that can acquire binding via proteolytic processing. Both forms of VEGF-B
bind to VEGF-receptor 1 (VEGFR-1) and the neuropilin-1 (NRP-1) coreceptor, which are expressed
mainly in blood vascular endothelial cells. VEGF-B-deficient mice and rats are viable without any
overt phenotype, and the ability of VEGF-B to induce angiogenesis in most tissues is weak. This has
been a puzzle, as the related placenta growth factor (PlGF) binds to the same receptors and
induces angiogenesis and arteriogenesis in a variety of tissues. However, it seems that VEGF-B is
a vascular growth factor that is more tissue specific and can have trophic and metabolic effects,
and its binding to VEGFR-1 shows subtle but important differences compared with that of PlGF.
VEGF-B has the potential to induce coronary vessel growth and cardiac hypertrophy, which can
protect the heart from ischemic damage as well as heart failure. In addition, VEGF-B is abundantly
expressed in tissues with highly active energy metabolism, where it could support significant
metabolic functions. VEGF-B also has a role in neuroprotection, but unlike other members of the
VEGF family, it does not have a clear role in tumor progression. Here we review what is hitherto
known about the functions of this growth factor in physiology and disease.
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INTRODUCTION
THE VASCULAR ENDOTHELIAL...
VEGF-B
SUMMARY
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I. INTRODUCTION
The blood circulatory system is the first organ system to
develop, starting in the third week of development in the
human embryo when passive diffusion of nutrients and
waste products becomes insufficient for development (118).
Members of the vascular endothelial growth factor (VEGF)
family are major regulators of blood and lymphatic vessel
development. Vasculogenesis, referring to the initial formation of the primitive vascular plexus, begins when hemangioblast progenitors of mesodermal origin migrate and differentiate to form primary blood islands, from which both
hematopoietic and endothelial cells are formed (114). The
primitive vascular plexus is subsequently remodeled into a
network consisting of arteries, veins, and capillaries of different sizes, and the vessels are stabilized by recruited mural
cells, such as pericytes and smooth muscle cells. Angiogenesis, the process of blood vessel formation from preexisting
vessels, occurs through either sprouting or intussusception
(splitting) in development and in various disease processes
(reviewed in Ref. 30; see also Ref. 113).
VEGF, the archetypal angiogenic growth factor, was first
identified as a permeability-inducing factor secreted by tumor cells (121) and later as a growth factor for vascular
endothelial cells (ECs) (46, 86). Other members of the
VEGF family were subsequently characterized, but VEGF-B
has remained one of the more ambiguous growth factors, as
efforts to discover angiogenic effects for it in vivo have
given mainly negative results. The aim of this review is to
summarize what is currently known about VEGF-B to endeavor to elucidate the main functions for this growth factor in physiology and disease. We first briefly review what is
known about the other members of the VEGF family and
their receptors before concentrating on VEGF-B.
II. THE VASCULAR ENDOTHELIAL GROWTH
FACTOR FAMILY
The VEGF family consists of five secreted dimeric glycoprotein growth factors in mammals: VEGF (or VEGF-A),
VEGF-B, VEGF-C, VEGF-D, and placenta growth factor
(PlGF) (for reviews, see Refs. 64, 125). As members of the
platelet-derived growth factor (PDGF)/VEGF superfamily,
VEGFs contain a VEGF/PDGF homology domain with
eight conserved cysteine residues involved in inter- and intramolecular disulfide bond formation. The VEGF ligands
bind with differing specificities to three mainly endothelial
transmembrane tyrosine kinase receptors, VEGFR-1/fms-like
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BRY ET AL.
tyrosine kinase 1 (Flt1), VEGFR-2/human kinase insert domain receptor (KDR)/mouse fetal liver kinase 1 (Flk1), and
VEGFR-3/fms-like tyrosine kinase 4 (Flt4) (for recent reviews,
see Refs. 77, 123) (FIGURE 1A). Neuropilins (NRP)-1 and
NRP-2, which transduce semaphorin signals mainly in neuronal cells, also provide coreceptor functions for the various
VEGFRs in endothelial cells (reviewed in Ref. 106).
which has hampered its application to therapeutic angiogenesis.
VEGF and VEGFR-1 are upregulated in hypoxia via hypoxia inducible factor (HIF)-1␣-mediated transcription (50,
110, 119). Nutrient and oxygen deprivation also induce the
potent metabolic regulator PGC-1␣ (peroxisome proliferator-activated receptor-␥ coactivator-1␣), which can induce
VEGF (and VEGF-B) independently of HIF-1␣ in skeletal
muscle (8, 18), highlighting the close coordination of blood
supply and regulation of tissue metabolism.
VEGF is essential for vasculature development, as mice lacking even a single VEGF allele die during embryogenesis as a
result of impaired angiogenesis and blood island formation
(23, 45). VEGF binds to VEGFR-1 and VEGFR-2 (35, 111), as
well as to NRP-1 and NRP-2 (55, 130).
PlGF was isolated from a human placental cDNA library
shortly after the discovery of VEGF (for a review, see Ref.
38). PlGF exists as four isoforms in humans, but only one
(PlGF-2) in mice. PlGF is expressed abundantly in the placenta, and at lower levels in for example the heart, lungs,
and skeletal muscle (for reviews, see Refs. 34, 38). PlGF is
very similar to VEGF-B in many respects, but their effects
on angiogenesis and arteriogenesis seem to differ considerably. PlGF binds to heparan sulfate and to the same receptors as VEGF-B, namely, VEGFR-1 and NRP-1 (95, 104).
PlGF gene-targeted mice are viable, but their angiogenesis
and arteriogenesis are impaired in ischemia, inflammation,
and wound healing, as well as in the hypoxic brain (24, 51).
PlGF deficiency was shown to impair adipose tissue growth
at least in part by inhibiting angiogenesis (90). Unlike
In addition to functioning as a mitogen for ECs, VEGF also
regulates endothelial cell survival (6, 15, 16). Downstream
signaling by VEGFR-2, following VEGF binding, receptor
dimerization, and autophosphorylation of several tyrosine
kinase residues, involves numerous pathways, including the
phospholipase C (PLC)-␥/protein kinase C (PKC) pathway,
resulting in the activation of the c-Raf-MEK-MAP kinase
cascade and cell proliferation (58, 135, 144). Activation of
the PI3K-Akt signal transduction pathway stimulates cell
survival and migration and eNOS activation (54). Administration of VEGF results in robust angiogenesis in various
tissues (reviewed in Ref. 44), but VEGF is also a potent
inducer of vascular permeability and inflammation (98),
A
PlGF
VEGF-B
VEGF-C
VEGF-D
VEGF
CCBE1+Protease
S S
S S
sVEGFR-1
NRP-1
NRP-1
VEGFR-1
NRP-2
VEGFR-2
VEGFR-3
B
1
2
3
4
5
6A 6B
Vegfb
7
exon
FIGURE 1. The VEGF family, receptor interactions, and structure of the VEGF-B
gene. A: specific binding of VEGFs to their
endothelial receptors. The dashed line indicates proteolytic processing that is required before VEGF-C and human VEGF-D
can bind to VEGFR-2. Proteolysis activates
VEGF-C and VEGF-D. Recent data show that
the collagen- and calcium-binding EGF domains 1 (CCBE1) protein promotes proVEGF-C binding to VEGFR-3 followed by proteolytic cleavage by a disintegrin and metalloprotease with thrombospondin motifs-3
(ADAMTS3), resulting in the mature form
of VEGF-C and increased VEGFR-3 signaling
(69a). B: schematic structure of the Vegfb
gene and its alternatively spliced transcripts. Shown are exons (numbered) and
introns with the alternative splice acceptor
(SA) sites that produce the VEGF-B167 and
VEGF-B186 isoforms, where exon 6A is lacking from VEGF-B167 mRNA. The arrowhead
indicates the site of proteolytic processing
of VEGF-B186. Red, sequence encoding the
VEGF homology domain.
167
SA: 186
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VEGF-B IN PHYSIOLOGY AND DISEASE
VEGF-B, PlGF is able to stimulate angiogenesis and collateral vessel growth in the ischemic heart and limb with similar efficiency as VEGF (92). PlGF also increases vessel permeability (20, 99) and inflammation (103, 120). However,
in a recent report, intracranial PlGF administration using an
adeno-associated virus (AAV) vector strongly stimulated
angiogenesis and arteriogenesis in the brain without significant inflammation or edema (52). PlGF has been reported
to stimulate the migration and survival of endothelial cells
(4, 153) and the proliferation of smooth muscle cells (13).
It has been reported in numerous studies that PlGF can make
heterodimers with VEGF, and some results have suggested
that it regulates intermolecular crosstalk between VEGFR-1
and VEGFR-2, in part via VEGF/PlGF heterodimer formation
(21, 104). However, PlGF also seems to be capable of inducing
unique signals downstream of VEGFR-1 compared with
VEGF and stronger VEGFR-1 tyrosine phosphorylation than
VEGF-B (7, 9, 82). PlGF may mediate most of its arteriogenic
effects via recruitment of growth factor-secreting monocytes
that express VEGFR-1 (108).
Much remains to be learned about the mechanisms of PlGFinduced vascularization. Interestingly, in the mouse heart,
PlGF was recently shown to induce myocardial angiogenesis and cardiac hypertrophy through a nitric oxide (NO)dependent mechanism via the Akt/mTORC1 pathway (69).
However, in another recent study, PlGF only secondarily
supported pressure overload-induced cardiac hypertrophy
through a paracrine mechanism via endothelial cells and
fibroblasts to induce capillary growth and fibroblast proliferation (3).
A. VEGFR-1
VEGFR-1 is composed of an extracellular ligand binding
part, a transmembrane domain, intracellular tyrosine kinase (TK) domain, and carboxy-terminal region (126). The
extracellular part consists of seven immunoglobulin-like
(Ig) domains. VEGF and PlGF bind to the second and third
Ig domains of VEGFR-1 and induce productive receptor
dimers for autophosphorylation in the TK domain (35,
136). Mice lacking VEGFR-1 produce an excess of ECs and
a disorganized vasculature and die in utero at E8.5–9 (48,
49). This suggests that at least during the development of
the vascular system, VEGFR-1 is mainly a negative regulator of angiogenesis because of its high affinity for VEGF, as
it can sequester VEGF away from actively signaling
VEGFR-2 homodimers. Similarly, VEGFR-1 deletion in
adult mice resulted in endothelial cell proliferation and increased vessel density in the lungs, heart, retina, brain, kidneys, and liver and protected against myocardial infarction
(MI), at least in part via upregulation of VEGFR-2 signaling
(63). This is consistent with the fact that mice engineered to
express only a truncated form of VEGFR-1 lacking the TK
domain responsible for signaling are viable and display no
obvious vascular defects (62).
The tyrosine kinase activity of VEGFR-1 in cultured endothelial cells is weak, and its downstream signaling is poorly
understood (reviewed in Ref. 124). On the other hand, in
addition to ECs, VEGFR-1 is expressed at least in monocytes/macrophages, and activation of its TK domain is required for their activation and migration in response to
ligand (11, 31). VEGFR-1 is also expressed in neuronal cells,
with implications in glial cell development and survival (28,
79, 143). A soluble form of the VEGFR-1 extracellular domain (sVEGFR-1), which is able to neutralize VEGF (74),
plays a role in regulating local guidance of emerging vessel
sprouts (26), and it has been shown to be involved in the
pathogenesis of preeclampsia (87, 105a, 112). Interestingly,
VEGFR-1 is expressed also in the endothelium of coronary
arteries in adult mouse and fetal human heart, whereas
VEGFR-2 is not, suggesting that VEGFR-1 could play a role in
coronary vessel development (70, 75, 105). VEGFR-1 mRNA
was also detected in cultured neonatal rat cardiomyocytes;
however, the expression level was three orders of magnitude
lower than in cultured endothelial cells (149). VEGFR-1 can
heterodimerize with VEGFR-2 and thus modify signaling
through VEGFR-2 homodimers (32). It is currently not
known if the expression pattern of VEGFR-1 is changed in
pathological conditions in, e.g., the heart or adipose tissue.
B. Neuropilins
The neuropilins, NRP-1 and NRP-2, were originally identified as receptors for semaphorins that mediate repulsive
signals during neuronal axon guidance (27, 78). NRP-1 and
NRP-2 lack cytoplasmic enzyme activity, but they function
as coreceptors, complexing with other transmembrane receptors such as the endothelial VEGFRs (reviewed in Refs.
106, 148). VEGF isoforms bind with differing specificities
to NRP-1 and NRP-2. NRP-1, which binds also VEGF-B
and PIGF (93, 95), is essential for the formation of the
vasculature, as mice lacking NRP-1 develop vascular defects and die at E13.5 (73). In the heart, NRP-1 is expressed
along with VEGFR-1 in coronary vessels, myocardial capillaries, and epicardial vessels (72, 105). Interestingly,
NRP-1 is also expressed in the developing myocardium and
endocardium in mouse embryos at E12.5 (93).
III. VEGF-B
A. Structure and Processing
VEGF-B (previously also known as VRF, VEGF-related factor), first discovered in 1996, has a structure very similar to
that of VEGF, and mouse VEGF-B shares ⬃43% amino
acid sequence identity with mouse VEGF164 (56, 101). The
Vegfb gene is highly conserved in mammals with ⬃88%
homology between the mouse and human growth factors at
the amino acid sequence level (101). A primitive form of
Vegfb with only 26% homology to human Vegfb may be
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BRY ET AL.
present in frogs, but Vegfb has not been identified in zebrafish (117). Vegfb consists of seven exons and generates
two isoforms because of the existence of alternative splice
acceptor sites in exon 6 (56, 102) (FIGURE 1B). VEGF-B167
has a highly basic heparin-binding carboxy terminus,
whereas VEGF-B186 contains a hydrophobic carboxy terminus and is modified by O-glycosylation and proteolytic
processing (102). VEGF-B167 thus binds tightly to heparan
sulfate proteoglycans on the cell surface and in the extracellular matrix, whereas at least the uncleaved VEGF-B186 is
freely diffusible. The molecular masses of homodimers of
VEGF-B167 and VEGF-B186 are 42 and 60 kDa, respectively. Both isoforms are simultaneously expressed in various tissues and bind to VEGFR-1 (and sVEGFR-1) as well
as to NRP-1, but not to the major mitogenic endothelial
receptors VEGFR-2 and VEGFR-3 (93, 100). Proteolytic
processing of VEGF-B186 is required for its binding to
NRP-1 (93). In culture, VEGF-B is also able to form heterodimers with VEGF (101), but covalent heterodimers of
this type have not been demonstrated in vivo.
Unlike VEGF, VEGF-B expression does not seem to be directly regulated by hypoxia (41), although hypoxia appeared to induce VEGF-B in the mouse retina in a recent
report (128). VEGF-B has a wide tissue distribution in mice,
being most abundant in tissues with high metabolic activity
such as the myocardium, skeletal muscle, and vascular
smooth muscle, as well as in brown adipose tissue, brain,
kidney, and parietal cells of the stomach (1, 22, 80, 101).
This suggests a role for VEGF-B in coordinating the crosstalk between angiogenesis and metabolism.
Analysis of VEGF-B mRNA in normal C57Bl/6J mouse tissues by quantitative RT-PCR confirmed that VEGF-B expression levels were the highest in the heart and skeletal
muscle (FIGURE 2A) (see also Ref. 59). Slow-twitch aerobic
muscles such as soleus and the red part of the gastrocnemius
had higher levels of VEGF-B than fast-twitch glycolytic
muscles, such as tibialis anterior and the white part of the
gastrocnemius, further supporting the idea that VEGF-B is
involved in aerobic energy metabolism. The levels of
VEGF-B in brown adipose tissue were between those found
in slow and fast muscles, while in visceral white adipose
tissue the levels were lower, although still higher than in
most other tissues. Staining of VEGF-B gene-targeted VegfblacZ/lacZ mouse hearts (14) for ␤-galactosidase shows
strong VEGF-B expression in the cardiomyocytes (FIGURE
2B).
Analysis of VEGF-B mRNA expression in healthy human tissues
using the MediSapiens database consisting of hundreds of microarrays of various human tissues (www.medisapiens.com) revealed the highest expression levels in the heart, adipose tissue,
skeletal muscle, and blood vessels, which also expressed high
levels of VEGFR-1 and NRP-1 (FIGURE 3, A AND B). The adipose
tissue samples from humans mainly represent biopsies from sub-
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cutaneous depots, which may have different expression levels
than visceral fat. Interestingly, VEGF-B and PlGF had very different expression patterns (FIGURE 3C). VEGF, which is required
during embryonic development, showed the broadest expression
across different tissues compared with VEGF-B and PlGF.
B. Differences Between VEGF-B and PlGF
Although VEGF-B and PlGF transduce their signals via
VEGFR-1 and NRP-1, their effects on blood vessel growth,
permeability, and tissue inflammation differ considerably
(20). However, both growth factors are dispensable for normal embryonic development.
Like many other receptor tyrosine kinases, the VEGFRs are
activated by ligand-induced dimerization, followed by tyrosine autophosphorylation of the intracellular kinase domain to generate downstream signaling (83). For ligand
binding, the VEGFRs share a conserved interface in Ig domain (D) 2, while D3 provides additional interactions (29,
67, 84, 85, 142). The membrane-proximal domains 4 –7
(D4 –7) facilitate dimerization through homotypic contacts
formed between D4 –5 and D7 (FIGURE 4A) (85, 116, 145).
VEGFRs show a hingelike rigid body motion in D2–3 (19,
84), suggesting that ligand-induced D2–3 reorientation initiates the cascade of structural rearrangements behind the
homotypic interactions and receptor activation.
Crystal structure analysis shows that VEGF-B, VEGF, and
PlGF interact with VEGFR-1 D2 in a similar manner (29,
67, 68, 142) (FIGURE 4B). VEGF and PlGF also require D3
of VEGFR-1 for high-affinity binding (29, 33). However,
D3 does not provide additional affinity for VEGF-B binding
to VEGFR-1 (7). The receptor specificity of VEGF ligands is
determined by an amino-terminal alpha helix and three
peptide loops. VEGF-B fails to efficiently induce productive
VEGFR-1 dimerization and signaling as a result of the
unique structure of VEGF-B loop 1 (L1; FIGURE 4, C AND
D). Importantly, swapping L1 from PlGF to VEGF-B conferred the angiogenic properties of PlGF to the resulting
chimera and vice versa. Since L1 in VEGFs interacts only
with D3 (19, 84), the poor ability of VEGF-B to activate
VEGFR-1 compared with PlGF can be explained by inadequate L1-D3 interactions. These results also suggest that, at
least in endothelial cells, the effects of VEGF-B occur
through inhibition of the interaction of other ligands with
VEGFR-1 (see also FIGURE 6), thus inducing more efficient
and spatially controlled interaction of VEGF with the
highly mitogenic VEGFR-2. How VEGF-B and PlGF signaling interactions are regulated in macrophages, which also
express VEGFR-1 and NRP-1 (25, 31), remains to be studied.
C. Role in Angiogenesis
Although initial reports indicated that VEGF-B is able to
stimulate EC growth in vitro (101), this may have been due
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VEGF-B IN PHYSIOLOGY AND DISEASE
A
1.2
mRNA expression of VEGF-B
relative to the heart (=1)
1
0.8
0.6
0.4
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as
ey
Pa
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ai
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ac
om
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β-gal
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WT
FIGURE 2. VEGF-B expression levels are the highest in the heart, skeletal muscle, and brown adipose tissue
in mice. A: quantitative RT-PCR of VEGF-B mRNA expression in 3-mo-old mouse tissues relative to the heart (set
to 1). Note that expression levels are higher in “red” muscles than in “white” muscles. Red and white GA
indicate red and white parts of gastrocnemius muscle. TA, tibialis anterior muscle; BAT, brown adipose tissue;
WAT, white adipose tissue. B: ␤-galactosidase (␤-gal) staining of gene-targeted VegfblacZ/lacZ mouse hearts
showing VEGF-B expression in the cardiomyocytes. WT, wild-type control heart. Scale bar: 50 ␮m.
to heterodimer formation with endogenous VEGF, as the
ability of recombinant VEGF-B to stimulate sprouting angiogenesis directly is poor in most tissues. VEGF-B did not
stimulate vessel growth when delivered into muscle or periadventitial tissue via adenoviral vectors (17, 115), and
transgenic overexpression of VEGF-B in the skin increased
blood vessel density only minimally, although it increased
capillary diameter to some extent (72). VEGF-B did not
improve vascular growth in the ischemic limb in two studies
(81, 88), although opposite results have also been published
(127, 140). On the other hand, VEGF-B overexpressed in
endothelial cells of transgenic mice was claimed to potenti-
ate, rather than initiate, angiogenesis (97). Overexpression
of VEGF-B was also shown to aggravate pathological retinal and choroidal neovascularization in mice (152) and was
proposed to act as a survival factor for ECs, regulating the
expression of vascular pro-survival genes via both NRP-1
and VEGFR-1 (150).
D. Effects of VEGF-B in the Heart
Mice deficient of VEGF-B are viable and fertile and display
only mild phenotypes in the heart. This is manifested as an
atrioventricular conduction abnormality characterized by a
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BRY ET AL.
A
2000
1500
1000
B
VEGF-B
VEGFR-1
NRP-1
C
PlGF
VEGF-B
Mesenchymal stem cell
Placenta
Kidney
Pancreas
Liver
Stomach
Adrenal gland
Pituitary gland
PNS ganglion
Cerebellum
Cerebrum
Lung
Skin
Connective tissue
Adipose tissue
Bone
Skeletal muscle
Heart
Blood vessel
Spleen
Lymph node
Hematopoietic stem cell
Reticulocyte
Blood dendritic cell
Blood monocyte
500
VEGF-A
Connective tissue
Placenta
Liver
Spleen
Kidney
Pancreas
Adrenal gland
Blood vessel
Mesenchymal stem cell
Adipose tissue
Heart
Skeletal muscle
Reticulocyte
Pituitary gland
Placenta
Adipose tissue
Heart
Mesenchymal stem cell
Kidney
Lymph node
Spleen
Adrenal gland
Blood vessel
Skeletal muscle
Blood dendritic cell
Pancreas
Connective tissue
Reticulocyte
Pituitary gland
FIGURE 3. Comparison of the RNA expression levels of VEGF-B and its receptors, PlGF and VEGF, in healthy
human tissues. A: box plot analysis of VEGF-B mRNA expression levels (arbitrary units) over a range of healthy
human tissues extracted from the MediSapiens database (http://www.medisapiens.com). Note the highest
VEGF-B expression levels in adipose tissue, heart, skeletal muscle, blood vessels, and reticulocytes. The y-axis
defines the expression level, while the x-axis shows the tissue type. B: comparison of the expression of VEGF-B
with its receptors, showing that among the tissues with high VEGF-B expression, adipose tissue, heart, skeletal
muscle, and blood vessels also highly express VEGFR-1 and NRP-1. C: comparison of the expression patterns
of VEGF-B and its closest relatives PlGF and VEGF. Note that the expression patterns of VEGF-B and PlGF, which
bind to the same receptors, are very different, whereas the metabolically active tissues (adipose tissue, heart,
and skeletal muscle) express abundant VEGF-B and VEGF. Darkest shades of blue indicate lowest relative
expression, and darkest shades of red indicate the highest relative expression.
prolonged PQ interval in one strain (2), or as a smaller heart
size with slightly dysfunctional coronary vasculature and
impaired recovery after myocardial ischemia in another
(14). The latter mouse strain also showed some resistance to
development of pulmonary hypertension and vascular remodeling during chronic hypoxia (141). VEGF-B-deficient
rats had no obvious vascular or developmental defects (75).
After experimental MI, larger infarct scars were detected in
cardiac sections from the VEGF-B gene-targeted rats, although heart function was not significantly affected (75).
Collectively, the results from VEGF-B gene-deleted mice
and rats suggest a role for VEGF-B in the heart in pathological settings.
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Interestingly, VEGF-B expression is spatially and temporally correlated with the commencement and progression
of coronary endothelial growth in the heart, suggesting
that it could play a role in coronary vessel development
(14). However, no obvious defects have been reported in
mouse embryos lacking VEGF-B. Importantly, however,
antibodies against VEGF-B were found to inhibit coronary artery development in the quail embryo (138, 139).
An excess of VEGF-B activates the Akt/mTORC1 and
Erk1/2 MAPK pathways in the heart (FIGURE 6) (75). Activation of Erk1/2 occurred at least in part through VEGFR-2
phosphorylation, an indirect result of VEGF-B binding to
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VEGF-B IN PHYSIOLOGY AND DISEASE
A
B
D2’
D1’
PIGF/VEGF-B
D2
D1
PIGF
D2
D2’
D3
D3’
VEGFR-1
C
αN
αN
D5’
L1
D5
L2
D
αN
D7’
D7
VEGFR ECD
L3
L1
L2
Variable
Conserved
FIGURE 4. Comparison of PlGF and VEGF-B structures. A: a model of the ligand-induced VEGFR extracellular
domain dimerization with the crystal structure of the PlGF/VEGFR-1 D2 complex (PDB code 1RV6; colored in
cyan) superimposed with a model of the VEGF-C/VEGFR-3 extracellular domain (ECD) complex (85). Ligandinduced homotypic interactions occur in the VEGFR membrane-proximal Ig-like domains D5 and D7 (85, 145).
B: PlGF and VEGF-B show similar modes of binding to VEGFR-1 D2 (29, 67). A top view is shown of the
superpositioning of the VEGF-B/VEGFR-1 D2 (PDB code 2XAC) and the PlGF/VEGFR-1 D2 complex structures
with VEGF-B and PlGF shown in ribbon diagram and colored in orange and cyan, respectively, and the VEGFR-1
D2 from the PlGF complex in cartoon and colored in gray. VEGFR-1 D2 from the VEGF-B complex is not shown.
C: the same as in B from a side view with the other PlGF monomer shown as a surface model. The
receptor-binding epitopes: loops 1 to 3 (L1 to L3) and the amino-terminal alpha-helix (␣N) are labeled. PlGF and
VEGF-B show a major difference in L1 and L3 conformations. D: a color-coded conservation pattern between
PlGF and VEGF-B in the VEGF homology region. A representative set of mammalian PlGF and VEGF-B amino acid
sequences were aligned using ClustalW, and the rate of conservation was calculated and plotted on PlGF in C
using Consurf server. Cyan through dark red is used for labeling from variable to conserved amino acids. As
shown by Anisimov et al. (7), PlGF and VEGF-B show significant differences in L1 and L3.
VEGFR-1, which increased the availability of VEGF for
activation of VEGFR-2 (75). The Erk1/2 activation via
VEGFR-2 is likely responsible for the coronary vessel
growth and arterialization observed in the VEGF-B overexpressing hearts. Importantly, blocking VEGFR-2 signaling
was able to reduce the vessel enlargement seen in VEGF-B
overexpressing mice. On the other hand, in contrast to the
hypertrophy induced by PlGF (69), blocking NO signaling
did not affect the VEGF-B-induced cardiac hypertrophy
(75). Importantly, the VEGF-B-induced hypertrophy remained physiological even in old rats, and did not advance
into heart failure (75).
E. VEGF-B as a Therapeutic Factor in
Cardiac Disease
Several studies have indicated a role for VEGF-B in coronary vasculature and in cardioprotection. VEGF-B levels
decreased following experimentally induced MI in rats as
well as in heart failure resulting from transverse aortic constriction (65, 151). Myocardial expression levels of
VEGF-B were decreased also in human heart disease (75).
Low VEGF-B plasma levels were found to accurately predict left ventricular dysfunction and remodeling after MI,
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suggesting that VEGF-B could be used as a prognostic biomarker with stronger predictive value than troponin T (36,
37). Interestingly, the opposite was true for PlGF, as increased levels of PlGF predicted heart failure (36). However, to date, studies about the kinetics of VEGF-B after
ischemic damage in the myocardium are lacking.
VEGF-B does not seem to induce capillary angiogenesis in
most studies, but mainly enlargement of myocardial capillaries and growth of coronary arteries (20, 72). In addition,
VEGF-B counteracts apoptosis of cardiomyocytes and improves cardiac contractility in animal models of human
heart disease (149). However, the responsible mechanisms
and signaling between endothelial cells and cardiomyocytes
in these settings are still largely unknown. Therapeutic vectors expressing VEGF-B have shown promise in several experimental models of myocardial ischemia or heart failure
(TABLE 1). An overdose of VEGF-B186 via transient adenoviral delivery into the pig myocardium enlarged myocardial
vessels after acute infarction, which was inhibited by administration of either soluble VEGFR-1 or soluble NRP-1,
but not by blocking VEGFR-2 signaling or nitric oxide production (81). Adenoviral delivery of VEGF-B167 activated
the PI3K/Akt pathway, enlarged capillaries and ameliorated angiotensin II-induced diastolic dysfunction in rats
(122). Adenoviral delivery of VEGF-B186 was equally capable of enlarging myocardial capillaries in mice (66). However, AAV-mediated administration of VEGF-B167 preserved cardiac contractility and prevented cardiomyocyte
apoptosis after experimental MI in rats without significant
vascular effects (149). Similar results were achieved in dogs
subjected to tachypacing-induced development of dilated
cardiomyopathy, where AAV-VEGF-B administration delayed the progression towards heart failure (107). In these
studies, it is important to note that adenoviral vectors me-
diate very high but transient levels of expression and induce
a strong inflammatory response, whereas AAVs mediate
steady, long-term expression without significant inflammation.
Transgenic overexpression of VEGF-B induced cardiac hypertrophy as well as capillary enlargement in both mice and
rats (20, 72, 88). In rats, VEGF-B also caused a striking
growth of the epicardial and subendocardial coronary vessels, which importantly occurred without increasing inflammation or vascular permeability, in contrast to other
members of the VEGF family (FIGURE 5) (20). The coronary
arterial growth induced by VEGF-B led to increased functional coronary reserve, protecting the heart from ischemic
damage caused by coronary artery ligation (75). Systemic
delivery of an AAV-VEGF-B vector to adult rats reproduced
both the vascular and hypertrophic phenotypes, an important consideration for possible therapeutic strategies. However, the effects of VEGF-B overexpression required 2–3
wk before becoming apparent, so it is unlikely that
VEGF-B induced vascular changes would be beneficial
during acute myocardial infarction. However, VEGF-B
could improve long-term recovery from ischemia and
counteract the development of heart failure. Indeed, a
VEGF-B186 adenovirus was shown to improve systolic
function in progressive left ventricular hypertrophy
caused by transverse aortic constriction in mice (65).
Thus the above studies and others have implicated the
heart as a specific target for VEGF-B.
F. Metabolic Effects of VEGF-B
Interestingly, expression of VEGF-B and a nuclear-encoded
mitochondrial gene and protein cluster set appear to be
Table 1. Experimental models of VEGF-B therapy in the heart
Vector/Transgene/Isoform
Species
Effect
Transgene (␣MHC-VEGF-B167)
Mouse
Transgene (␣MHC-genomic VEGF-B)
Rat
Adenovirus (VEGF-B186)
Adenovirus (VEGF-B186)
Adenovirus (VEGF-B167)
Pig
Mouse
Rat
Cardiomyocyte hypertrophy; myocardial capillary enlargment; ischemia protection
ex vivo (72)
Cardiomyocyte hypertrophy; myocardial capillary enlargement; expansion of
coronary arteries; no increased inflammation or vascular leakage (20); increased
functional coronary reserve; ischemia protection in vivo (75)
Enlargement of myocardial vessels after myocardial infarction (81)
Capillary enlargement (66)
Capillary enlargement and improved cardiac function in angiotensin II-induced
diastolic dysfunction (122)
Increased density of capillaries and arterioles in infarct zone (88)
Increased capillary density and cardiomyocyte area in remote myocardium after
ischemia (137)
Antiapoptotic effect on cardiomyocytes after myocardial infarction;
cardiomyocyte hypertrophy; no significant vascular effects (149)
Delayed progression of heart failure during tachypacing-induced cardiomyopathy;
antiapoptotic effect on cardiomyocytes (107)
Improves systolic function following transverse aortic constriction; antiapoptotic
effect on cardiomyocytes; capillary enlargement (65)
Adenovirus or protein infusion (VEGF-B167) Mouse
Adenovirus (human VEGF-B167)
Mouse
AAV2-VEGF-B167
Rat
AAV9-VEGF-B167
Dog
AAV9-VEGF-B186
Mouse
786
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VEGF-B IN PHYSIOLOGY AND DISEASE
A
WT
LV
B
WT
coordinately regulated, which is not the case for the other
VEGF family members (59, 96). VEGF-B expression in skeletal muscle is upregulated by the transcription factor
PGC-1␣ (18, 134), as has been previously shown for VEGF
(8). In addition, VEGF-B expression was induced in skeletal
muscle by voluntary exercise in both mice and humans (18,
76). As PGC-1␣ is the major regulator of mitochondrial
biogenesis and oxidative metabolism, these findings underline the close interplay between oxygen delivery by the vasculature and target tissue metabolism.
VEGF-B deletion was recently reported to lead to decreased expression of fatty acid transport proteins
(FATPs) FATP-3 and FATP-4 in endothelial cells. This
correlated with a decreased amount of lipid droplets in
cardiomyocytes and skeletal muscle fibers, leading to increased white adipose tissue mass and body weight in old
mice (59). Further analysis demonstrated that VEGF-Bdeficient mice fed a high-fat diet or crossed with diabetic
db/db mice have improved insulin sensitivity, glucose homeostasis, and blood lipid profiles (60). This was accompanied by reduced lipid storage in muscle, heart, and
TG
LV
TG
FIGURE 5. Cardiac arteriogenesis in VEGF-B
transgenic rats. A myocardium-specific VEGF-B
transgene induced impressive growth of coronary
arteries and their branches in rats, without increasing inflammation or vascular leakage, as
shown by Bry et al. (20) and Kivelä et al. (75).
A: shown are representative immunofluorescence
images of capillaries (green) and smooth muscle
cells (yellow) in the myocardium of a VEGF-B transgenic (TG) and control (WT) rat. LV indicates the
left ventricle. Scale bar: 200 ␮m. B: 3-dimensional micro-computed tomography (CT) images
of the coronary artery tree in WT and VEGF-B TG
rats.
pancreas, but not in the liver, and increased weight gain
on high-fat diet. VEGFR-1 and its tyrosine kinase activity
as well as NRP-1 were needed for the VEGF-B-mediated
effects on fatty acid transport across the endothelium
(59). These results suggested that endothelial growth factors play a role in tissue lipid homeostasis and could
provide a promising novel target for the treatment of
metabolic and cardiovascular diseases.
The functional importance of angiogenesis and vascular
endothelial regulation in adipose tissues has gained increased attention recently. Interestingly, both repression
(91) and overexpression (40, 131) of VEGF were reported
to protect against high-fat diet-induced obesity and/or insulin resistance. The expression of VEGF-B and FATPs 1– 4
were increased in white adipose tissue of VEGF-deficient
mice, via an unknown mechanism (91). These mice demonstrated lower body weight and resistance to high-fat dietinduced obesity, together with upregulation of brown adipose tissue-specific genes in white adipose tissue. In another
study, the expression of VEGF-B and FATP-3 in brown
adipose tissue was reduced after cold exposure, but mech-
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BRY ET AL.
anistic experiments were not conducted (12). VEGF-B was
also shown to affect FATP-1 and FATP-4 levels in a neuroblastoma cell line (147).
In rats, however, we found no difference in fatty acid uptake
between VEGF-B transgenic, gene-deleted, or wild-type
hearts, although VEGF-B administration increased the levels
of FATP-4 mRNA (75). In fact, fatty acid and triglyceride
levels were actually reduced in the rat hearts overexpressing
VEGF-B. On the other hand, VEGF-B overexpression led to
an adjustment of cardiomyocyte metabolic pathways in favor
of glucose oxidation and macromolecular biosynthesis. These
metabolic changes may have contributed to the ischemia protection seen in this model, where cardiomyocyte mitochondria
were protected against ischemia-reperfusion injury (75). Thus
it is clear that the metabolic effects of VEGF-B are not as
simple as has been previously suggested, and much remains to
be learned about the specific role of VEGF-B in metabolic
regulation.
In summary, there are several studies describing the role of
vascular growth factors in adipose tissue homeostasis.
Some studies have reported changes in VEGF-B expression
by therapeutic drugs, such as rosiglitazone, which stimulates peroxisome proliferator-associated receptor-␥ (53),
but so far no study has investigated the role of VEGF-B in
adipose tissue regulation. Based on the gene expression data
from both mice and humans, VEGF-B and its receptors are
highly expressed in both white and brown adipose tissue,
suggesting that VEGF-B could have a function also in these
tissues. An important question is whether VEGF-B acts in a
paracrine manner via the endothelium, directly on the adipocytes, or also via VEGFR-1 expressing macrophages,
which are important in adipose tissue regulation especially
in pathological settings (94).
G. VEGF-B-Induced Neuroprotection
VEGF-B is also expressed in the central nervous system (1).
Interestingly, VEGF-B-deficient mice showed impaired recovery from cerebral ischemic injury (132), and administration of VEGF-B stimulated neurogenesis in adult mice
(133). Furthermore, VEGF-B186 protected primary motor
neurons against degeneration in culture (109). Accordingly,
mice lacking VEGF-B developed a more severe form of motor neuron degeneration when crossed with SOD1 mutant
mice, a model of amyotrophic lateral sclerosis. Importantly,
administration of VEGF-B186 into the ventricles of the brain
prolonged the survival of mutant SOD1 rats, and the in
vitro and in vivo effects of VEGF-B were dependent on the
tyrosine kinase activity of its receptor VEGFR-1 in motor
neurons.
VEGF-B was furthermore upregulated in an in vitro model
of Parkinson’s disease induced by the neurotoxin rotenone,
and the addition of exogenous VEGF-B167 improved neu-
788
ronal survival (43). In a preclinical in vivo Parkinson’s disease model, exogenous VEGF-B186 showed neuroprotective
effects, likely due to the partial protection of dopaminergic
fibers in the striatum and partial rescue of the dopaminergic
neurons in the caudal subregion of the substantia nigra
(42). This neuroprotective effect was further investigated in
a follow-up study, where VEGF-B expression was confirmed in the substantia nigra pars compacta of human
patients with Parkinson’s disease (147).
VEGF-B and VEGFR-1 are expressed in dorsal root ganglion neurons that innervate the hindlimb skin (39). Mice
lacking VEGF-B or functional VEGFR-1 developed more
retrograde degeneration of sensory neurons in a distal neuropathy model, whereas mice overexpressing VEGF-B186 or
VEGFR-1 selectively in neurons were protected against the
distal neuropathy. Exogenous VEGF-B186 was shown to be
protective by directly affecting sensory neurons and not the
surrounding vasculature (39). Inhibition of apoptosis was
suggested as a possible mechanism for the VEGF-B-induced
neuroprotection (89). VEGF-B treatment also rescued neurons from apoptosis in the retina and brain in mouse models
of ocular neurodegenerative disorders and stroke, respectively.
The neuroprotective effect of VEGF-B seems to be directly
mediated by VEGFR-1 expressed by the neuronal cells and
not via improved angiogenesis (39, 109). As a possible protection mechanism, inhibition of apoptosis by VEGF-B has
also been proposed in cardiac ischemia (149). However,
further studies are needed to clarify this as a potential
downstream mechanism of VEGF-B - VEGFR-1 signaling.
H. VEGF-B in Cancer
It has been proposed that VEGF-B could play a role early in
tumor development at the stage of adenoma formation, and
VEGF-B expression is upregulated in for example ovarian,
colorectal, renal, and prostate cancer (57, 61). However,
only one study has addressed the role of VEGF-B in tumor
development using a genetic model (5). This study analyzed
the effects of VEGF-B overexpression versus deficiency in
the context of pancreatic endocrine adenocarcinoma in
transgenic RIP1-Tag2 mice. Interestingly, ectopic expression of VEGF-B reduced tumor growth, whereas mice lacking VEGF-B presented with larger tumors. The mechanism
of VEGF-B action, however, remained unsolved, but the
tumor vasculature and inflammation, the usual suspects,
were not affected by the presence or absence of VEGF-B.
The role of PlGF in tumor angiogenesis is also controversial
(10, 47, 146). PlGF blocking antibodies were reported to
inhibit the growth of medulloblastomas and tumors that
express VEGFR-1 in tumor cells in addition to endothelial
cells (129, 146). However, it is not known whether the
expression levels of VEGF-B in the tumors affect the efficacy
of anti-PlGF therapies.
Physiol Rev • VOL 94 • JULY 2014 • www.prv.org
VEGF-B IN PHYSIOLOGY AND DISEASE
IV. SUMMARY
Because VEGF-B is dispensable during embryonic development and even high VEGF-B expression levels induced
by viral delivery do not induce adverse effects, such as
inflammation or vascular leakage, VEGF-B seems to possess a wide therapeutic window, which would be important for its possible use in human diseases. The idea that
a vascular growth factor could also regulate tissue metabolism may sound unexpected at first; however, it is
logical that both oxygen delivery by blood vessels and
oxygen usage by mitochondria would be coordinated to
be able to match nutrient/oxygen supply and demand.
The close interplay between angiogenesis and mitochondrial biogenesis and oxidative metabolism will likely be
the focus of numerous studies in the near future, and
VEGF-B is one potential candidate molecule in this crosstalk.
VEGF-B still remains an enigmatic vascular growth factor,
but recent studies have indicated that VEGF-B can regulate
blood vessel and cardiomyocyte growth in the heart, tissue
metabolism, and cell survival (FIGURE 6). In addition to its
effects on endothelial cells, studies in the central nervous
system provided evidence that VEGF-B can directly affect
neurons expressing VEGFR-1. Overall, VEGF-B seems to
be important for tissue protection, whether it be in the
context of heart failure or neuron degeneration. The possible role of VEGF-B in coronary artery development is an
open question, but since gene-deleted mice and rats are
viable and fertile and born in normal Mendelian ratios, this
has not been studied in detail.
EC
VEGF-B
VEGFR-1
VEGF
NRP-1
PF
PF
O2 and
Nutrients
VEGFR-1
VEGFR-2
VESSEL SIZE
ARTERIALIZATION
pERK
pERK , pAkt
APOPTOSIS
pAMPK
mTOR
Malonyl-CoA
pS6K1
FASN
pS6
FATTY ACID SYNTHESIS
HYPERTROPHY
PROTEIN SYNTHESIS
CMC
FIGURE 6. A schematic model of the main effects of VEGF-B overexpression in the heart. VEGF-B, expressed
by cardiomyocytes, binds to VEGFR-1 expressed in vascular endothelial cells and NRP-1 expressed in both
endothelial cells and cardiomyocytes. Elevated levels of VEGF-B can displace VEGF from VEGFR-1, therefore
increasing VEGF availability for VEGFR-2, which can induce capillary growth and arteriogenesis via increased
Erk1/2 signals (9, 63, 75). Endothelial-cardiomyocyte crosstalk, which employs paracrine factors (PF), matrix
molecules, and small-molecular-weight components, is essential for the coordination of physiological heart
adaptation to various stresses. The vascular endothelium is also central as a gateway for the transport of
nutrients and oxygen to the cardiomyocytes. Shown in the lower part of the figure are some of the signaling
pathways and downstream effects of VEGF-B overexpression in the heart, leading to increased fatty acid and
protein synthesis, cardiomyocyte hypertrophy, and decreased apoptosis (75, 107, 122, 149). AMPK, AMPactivated protein kinase; FASN, fatty acid synthase; mTOR, mammalian target of rapamycin; S6K, ribosomal
protein S6 kinase.
Physiol Rev • VOL 94 • JULY 2014 • www.prv.org
789
BRY ET AL.
ACKNOWLEDGMENTS
pendent regulation of VEGF and angiogenesis by the transcriptional coactivator PGC1alpha. Nature 451: 1008 –1012, 2008.
We thank Drs. Seppo Ylä-Herttuala and Ulf Eriksson for
collaboration; however, the views presented in this review
are the sole responsibility of the authors. We apologize that
a number of valuable contributions could not be cited because of space restrictions.
M. Bry and R. Kivelä contributed equally to this article.
Address for reprint requests and other correspondence:
K. Alitalo, Wihuri Research Institute, Biomedicum Helsinki, PO Box 63 (Haartmaninkatu 8), FI-00014 University of Helsinki, Helsinki, Finland (e-mail: kari.alitalo@
helsinki.fi).
GRANTS
The authors’ studies were supported by Academy of Finland Grants 265982, 272683, 273612, and 273817; the
Sigrid Juselius Foundation; European Research Council
Grant ERC-2010-AdG-268804, Marie Curie Actions FP7/
2007-2013REA 317250; Leducq Foundation Grant
11CVD03; and the Finnish Foundation for Cardiovascular
Research.
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No conflicts of interest, financial or otherwise, are declared
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