Activated forms of VEGF-C and VEGF-D provide improved
vascular function in skeletal muscle
Andrey Anisimov
1
, Annamari Alitalo
2
, Petra Korpisalo
2
, Jarkko Soronen
1
, Seppo
Kaijalainen
1
, Veli-Matti Leppänen
1
, Michael Jeltsch
1
, Seppo Ylä-Herttuala
2
, and Kari
Alitalo
1
1
Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, Department of Pathology, Haartman
Institute and Helsinki University Central Hospital, P.O.B. 63, (Haartmaninkatu 8), 00014 University
of Helsinki, Finland
2
Department of Biotechnology and Molecular Medicine, A. I. Virtanen Institute
for Molecular Sciences, University of Kuopio, P.O.B. 1627, 70211 Kuopio, Finland
Abstract
The therapeutic potential of vascular endothelial growth factor C (VEGF-C) and VEGF-D in skeletal
muscle has been of considerable interest as these factors have both angiogenic and lymphangiogenic
activities. Previous studies have mainly employed adenoviral gene delivery for short-term expression
of VEGF-C and VEGF-D in pig, rabbit and mouse skeletal muscles. Here we have used the activated
mature forms of VEGF-C and VEGF-D expressed via recombinant adeno-associated virus (rAAV),
which provides stable, long-lasting transgene expression in various tissues including skeletal muscle.
Mouse tibialis anterior muscle was transduced with rAAV encoding human or mouse VEGF-C or
VEGF-D. Two weeks later, immunohistochemical analysis showed increased numbers of both blood
and lymph vessels, and doppler ultrasound analysis indicated increased blood vessel perfusion. The
lymphatic vessels further increased at the four-week time point were functional, as shown by FITC-
lectin uptake and transport. Furthermore, receptor activation and arteriogenic activity were increased
by an alanine substitution mutant of human VEGF-C (C137A) having an increased dimer stability
and by a chimeric CAC growth factor that contained the VEGF receptor-binding domain flanked by
VEGF-C propeptides, but only the latter promoted significantly more blood vessel perfusion when
compared to the other growth factors studied. We conclude that long-term expression of VEGF-C
and VEGF-D in skeletal muscle results in the generation of new functional blood and lymphatic
vessels. The therapeutic value of intramuscular lymph vessels in draining tissue edema and
lymphedema can now be evaluated using this model system.
Keywords
VEGF-C; VEGF-D; adeno-associated virus; angiogenesis; lymphangiogenesis; skeletal muscle
Introduction
Blood and lymphatic vessels integrate to form the circulatory system, which provides oxygen
and nutrients to the tissues and removes carbon dioxide and waste metabolites. Endothelial
Correspondence to Kari Alitalo, MD, PhD, Molecular/Cancer Biology Laboratory, Biomedicum Helsinki, P.O.B. 63, (Haartmaninkatu
8), University of Helsinki, FI-00014, Helsinki, Finland; Phone: 358-9-1912 5511; Fax: 358-9-1912 5510; Kari.Alitalo@Helsinki.FI.
Disclosures
K.A. is the Chairman of VeGenics Scientific Advisory Board
NIH Public Access
Author Manuscript
Circ Res. Author manuscript; available in PMC 2010 June 5.
Published in final edited form as:
Circ Res. 2009 June 5; 104(11): 1302–1312. doi:10.1161/CIRCRESAHA.109.197830.
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cells (ECs) form a surface barrier between the intra-and extravascular spaces, and EC number
determines the functional capacity of the vasculature in each tissue. Vascular endothelial
growth factors (VEGFs) are considered to be the main mitogenic and survival factors for ECs,
obligatory for embryonic angio- and lymphangiogenesis and capable of activating ECs in the
adult.
1–3
In mammals, the five known VEGFs are VEGF-A, B, C, D and placenta growth factor
(PlGF). Of these, VEGF-C and VEGF-D activate primarily lymphatic ECs, which express
VEGF receptor (VEGFR)-3.
4,5
After biosynthesis, full-length (fl) VEGF-C and VEGF-D
undergo proteolytic cleavage of their C- and N-terminal propeptide domains.
6,7
This releases
the mature growth factor (ΔNΔC, here short form - sf) with an increased affinity towards
VEGFR-3 and towards VEGFR-2, which is expressed mainly in blood vessels. VEGFR-2
binding has been assumed to be responsible for the angiogenic properties of VEGF-C and
VEGF-D in a number of experimental conditions.
8–10
However, no systematic studies have
addressed the capacity of the proteolytically activated, mature VEGF-C/D forms to stimulate
angiogenesis vs. lymphangiogenesis in vivo.
The ability to stimulate both VEGFR-2 and VEGFR-3 in blood and lymphatic vessels has made
VEGF-C and VEGF-D attractive candidates for therapeutic improvement in many types of
vascular insufficiencies, including skeletal and cardiac muscle ischemia, lymphedema, and
lymphatic vessel hypoplasia.
11
Adenoviral gene transfer was effective in delivering vascular
growth factors to pig myocardium,
12
rabbit and mouse skeletal muscle,
9,13
and to the
periadventitial tissue of rabbit carotid arteries.
14
Transgene expression was found to be robust,
but transient, lasting only one to three weeks, yet it resulted in significant improvement of
tissue perfusion and capillary size. However, declining levels of transgene expression lead to
significantly decreased perfusion and to pruning of newly formed blood vessels.
9,12
In contrast
to adenovirus, recombinant adeno-associated virus (rAAV) is an effective vehicle for
delivering transgenes into many types of tissues and organs including muscle, liver, and brain.
15,16
While rAAV transgene expression levels are lower compared to adenovirus, they are
permanent in the muscle.
17–19
This feature of rAAV may provide a vessel maintenance
function when vascular growth factors are expressed. These properties of the rAAV vectors
prompted us to study the long-term angiogenic vs. lymphangiogenic properties of the full-
length vs. short forms of VEGF-C, VEGF-D and, for comparison, VEGF-A or a chimeric CAC
growth factor that contained the VEGF receptor-binding domain flanked by VEGF-C
propeptides, all delivered via rAAV into skeletal muscle of mice. We addressed in particular
the effects of the activated, mature short forms of VEGF-C and VEGF-D.
Materials and Methods
Expanded Materials and Methods section containing detailed description of cell survival assay
and analysis of protein expression, rAAV vector preparation, protein purification, isothermal
titration calorimetry, VEGFR-2 and VEGFR-3 ELISA assays, immunohistochemistry,
Doppler ultrasound measurements of perfusion in the transduced muscles and FITC-lectin
microlymphography are provided as supplemental material.
Muscle transduction by the rAAV vectors
Six to seven week-old female FVB/NJ, ICR and C57BL/6J mice (three to four per group) were
anesthetized with xylazine (Rompun, Bayer)-ketamine (Ketalar, Pfizer), and 5 × 10
10
rAAV
particles (in 30 μl volume) were injected into each tibialis anterior (t.a.) muscle. All mouse
experiments were approved by the Provincial State Office of Southern Finland and carried out
in accordance with institutional guidelines.
Anisimov et al. Page 2
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Fluorescent angiography of blood vessels via cardiac perfusion
Blood vessels were directly visualized by cardiac perfusion with the lipophilic carbocyanine
dye 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI)
20
. After
perfusion, the tibialis anterior muscle was isolated and 120 μm sections were analysed by
confocal microscopy using the excitation wavelength of 543 nm.
Statistical analysis
We evaluated statistical significance by analysis of variance using the Dunnett (2-sided) test
as a post-hoc test, with P<0.05 regarded as significant. Where pairwise comparisons between
experimental groups were made, the Tukey test was used. Where indicated, Student’s t-test
was also used. The results are presented as mean values ±SD, unless otherwise indicated. All
experiments were reproduced at least three times.
Results
Expression and analysis of recombinant VEGF-C and VEGF-D
The regions encoding the proteolytically processed mature forms of human and mouse VEGF-
C and VEGF-D, denoted VEGF-Csf and VEGF-Dsf, were cloned downstream of a signal
sequence placed under the CMV promoter in the rAAV8 vector. Figure 1A shows the amino
acid sequences of human and mouse VEGF-Csf. The residues that differ between the mouse
and human sequences are marked in blue and green (alignment of sequences from seven animal
species is shown in Online Figure I). Two residues (valine-146 and serine-179) in hVEGF-Csf
were substituted to alanine (V146A) and glycine (S179G), respectively, that are found in the
mVEGF-Csf protein. Alternatively, the cysteine-137 residue in hVEGF-Csf (marked red) that
is not conserved in VEGF-A, VEGF-B or PlGF, was substituted to alanine.
Production and receptor binding of the vector-encoded growth factors was analyzed from the
medium of transfected 293T cells, precipitated with VEGFR-2-Ig or VEGFR-3-Ig (Figure 1B).
The full-length cDNAs were also expressed and their products migrated as full-length,
partially-processed and fully processed polypeptides (Figure 1C), as described previously.
6,7
Note that the VEGF-Dsf and VEGF-Dfl bands appear relatively weaker when compared to the
corresponding VEGF-C bands. This may reflect their significantly lower affinity to the
receptors used for precipitation.
2
VEGF-C137A provides increased dimer stability and receptor activity
Prior to the in vivo experiments, recombinant proteins were assayed for stimulation of receptor
dimerization-mediated cell proliferation using mouse BaF3 pro-B cells expressing either
VEGFR-2-Epo or VEGFR-3-Epo chimeric receptor (see supplemental Materials and Methods
for details). Dilutions of transfected 293T cell media containing similar amounts of the growth
factors were used. While the full-length growth factors were processed to several forms in the
conditioned medium, the short forms had increased activity (see asterisks and brackets in
Online Figure II). Overall, the results also indicated that VEGF-D is less active than VEGF-C
in stimulating VEGFR-3 and especially VEGFR-2 activation (Online Figure II). These results
are consistent with previously published data on VEGFR-2/VEGF-D interaction
2,5,21
and with
the data reported for phosphorylation of VEGFR-2 stably expressed by porcine aortic
endothelial cells.
22
Of all the mutant human VEGF-C:s, the strongest stimulation of BaF3 cell proliferation was
obtained with hVEGF-Csf C137A (Figure 1D and E). Compared to the parental growth factor
(hVEGF-Csf), this protein was particularly active towards VEGFR-2/BaF3 cells (Figure 1D).
The G175 residue in the mouse sequence also seemed to provide increased activity over the
human S179 (hVEGF-Csf S179G vs. hVEGF-Csf in Figure 1D), whereas replacement of V146
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of hVEGF-Csf to A (corresponding to A142 in mouse VEGF-C) resulted in unaltered or
diminished activity (Figure 1D, E).
According to a model based on the structures of the other VEGF family proteins
23–25
, we
predicted that the location of the C137 residue is in the dimer interface of hVEGF-Csf (Figure
2A). Despite its increased activity in BaF3 cell proliferation assays, isothermal titration
calorimetry indicated that hVEGF-Csf C137A binds VEGFR-3 with an affinity similar to that
of the wild-type protein (Kd 280 ± 30 nM and Kd 310 ± 50 nM, respectively; Figure 2B).
Further analysis employed an ELISA-based competition assay, where plates were coated with
VEGF
165
(for VEGFR-2 binding) or hVEGF-Csf (for VEGFR-3) and then incubated with
VEGFR-2-Ig or VEGFR-3-Ig proteins preincubated with dilutions of a blocking hVEGF-Csf
or its C137A mutant. The amounts of receptor fusion proteins bound to the plates were then
quantified by using HRP-conjugated antibodies. The results indicated that wild-type and
mutant hVEGF-Csf do not differ significantly in binding to VEGFR-2 (EC
50
2.6 ± 0.3 and 2.7
± 0.6 nM, respectively) or VEGFR-3 (EC
50
2.2 ± 0.6 and 1.3 ± 0.3 nM, respectively; P>0.05
for both comparisons, Student’s t-test) (Figure 2C). However, a difference was obtained in
non-reducing conditions where hVEGF-Csf C137A migrated as a dimer, while the other two
hVEGF-Csf mutants and the wild-type protein also contained some monomers. When treated
with the reducing agents 2-ME or DTT (Figure 2D,E), hVEGF-Csf C137A retained a dimeric
structure in conditions in which the native hVEGF-Csf protein was monomeric (1 mM DTT).
Thus, the increased activity of the hVEGF-Csf C137A mutant protein in the cellular bioassay
probably resulted from an increased stability of its dimeric structure.
rAAV8-VEGF-C/D transgenes induce both angiogenesis and lymphangiogenesis
The plasmid vectors used in the in vitro studies were subsequently packaged into rAAV8 and
injected into mouse t.a. muscle. Two or four weeks after the injection, the muscles were
processed for immunohistochemical staining of PECAM-1 (endothelial cells), MECA32
(blood vascular endothelial cells only), LYVE-1 (lymphatic endothelial cells), SMA (smooth
muscle cells and pericytes) and CD45 (leukocytes). Additional immunostaining was done for
PROX-1 and VEGFR-3 to confirm the lymphatic vessel phenotype. Three different mouse
strains (FvB/NJ, C57Bl/6J and ICR) were used in the experiments to validate the key findings
as several studies have demonstrated significant interstrain differences in their responses to the
angiogenic growth factors bFGF and VEGF.
26,27
In muscles of the FvB/NJ and ICR mice, only small changes were observed in the blood or
lymphatic vessels two weeks after injection of the vectors (data not shown). Analysis at four
weeks, however, revealed both an angiogenic and a lymphangiogenic response to the short
forms of the factors (Figure 3; Online Figure III, Online Figure IV). Double immunostaining
of PECAM-1 and mVEGF-Dsf suggested a paracrine vascular effect around the growth factor
producing myofibers (Online Figure V). Full-length mVEGF-C induced only some
lymphangiogenesis, but no angiogenesis, while mVEGF-Dfl had no effect. The
lymphangiogenic response was confirmed in parallel samples stained for PROX-1 (data not
shown). Blood vessels (MECA32) with their associated perivascular smooth muscle (SMA)
were notably increased in skeletal muscles injected with rAAV-mVEGF-Csf, which was also
the most active among the wild-type factors in the VEGFR-2/BaF3 cellular assay (Figure 1
and Online Figure II). rAAV-VEGF
165
, used as a control, induced only angiogenesis (Figure
3).
The vascular response in C57Bl/6J skeletal muscle was more rapid, being clear by two weeks
after the injection of the vectors. As all mice of this strain injected with the corresponding
concentrations of the control rAAV8-VEGF
165
vector died or had to be euthanized (due to
VEGF
165
-induced vascular leakage
28
), we instead used a chimeric VEGF-A called CAC that
contains the VEGF
165
residues 37 to 135 (VEGF homology domain) flanked with the
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propeptides of VEGF-C
29
. The mice tolerated treatment with this factor without overt
symptoms despite the fact that rAAV-CAC induced significantly stronger angiogenic and
arteriogenic responses than hVEGF-Csf, hVEGF-Dsf or mVEGF-Dsf in the skeletal muscles
(MECA32 and SMA panels in Figure 4A). A vector encoding the hVEGF-Csf C137A mutant
was also tested. Its arteriogenic effects were significantly stronger when compared to the
parental factor hVEGF-Csf (Figure 4A), although its lymphangiogenic activity was not
significantly different (see the LYVE-1 panel in Figure 4A: P>0.05, when hCsf137A is
compared to any other experimental group). These effects were even more enhanced at week
11, the latest time point analyzed (data not shown). In all muscle samples analyzed, we observed
a strong accumulation of CD45 positive cells with a tendency to concentrate in areas of
neovesssel formation (Figure 4A and data not shown).
Increased blood vessel perfusion in the muscles expressing VEGF-C/D
In order to observe any changes in blood perfusion in the treated muscles, we used Doppler
ultrasound analysis to detect functional blood vessels with diameters of 30 μm or more. In
C57Bl/6J skeletal muscles treated for two weeks with the various factors, significantly
increased perfusion was recorded (Figure 5). The strongest effect was observed in muscles
treated with rAAV-CAC (p<0.05). rAAV-hVEGF-C C137A also showed a strong angiogenic
effect, although not statistically different from the effects of wild-type VEGF-C/Dsf proteins.
The effect of mVEGF-Dsf seemed to be as strong as that of mVEGF-Csf, whereas the former
was significantly weaker in inducing SMA-positive perivascular cells (Figure 4A).
In order to further image the morphology and perfusion of vessels, the lipophilic dye DiI was
injected to the left ventricle of the heart. This fluorescent dye is incorporated into blood vascular
endothelial cell membranes, while unbound dye in the vessel lumen is washed away by
subsequent perfusion-fixation.
20
Confocal microscopy of thick muscle sections demonstrated
that the AAV mediated VEGF-C/D expression leads to widening of preexisting vessels (Figure
6). Increased neovessel formation by sprouting was also observed in the hVEGF-Csf 137A
treated samples and in the positive control (CAC). The CAC induced vessels differed greatly
from those induced by VEGF which resulted in local angioma-like vascular patterns in the
treated muscles (compare CAC and VEGF in Figure 6).
VEGF-C and VEGF-D induced intramuscular lymphatic vessels are functional
Comparison of MECA32, LYVE-1 and PROX-1 immunostaining after four weeks of transgene
expression in FvB/NJ mice demonstrated that the majority of the induced endothelium within
the muscles was of lymphatic origin (Figure 3 and data not shown). In comparison, neither
VEGF
165
nor CAC induced lymphatic vessel growth (Figures 3 and 4).
We used FITC-conjugated Lycopersicon esculentum lectin
30,31
to determine if the newly
formed lymphatic vessels were functional, i.e. capable of absorbing macromolecules (such as
FITC-lectin) and conducting fluid flow. Indeed, FITC-lectin injected to the distal end of the
t.a. muscle was uniformly distributed within the newly formed lymphatic vessels 45 min after
injection, although some was also present in between the myofibers (Figure 7), demonstrating
that the vessels were functional.
Discussion
We show here that rAAV-delivered activated forms of VEGF-C and VEGF-D induce both
angiogenesis and lymphangiogenesis in skeletal muscle and that the latter response tends to
predominate at the four-week time-point used in our experiments.
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Previously, adenovirally delivered human VEGF-D
ΔNΔC
or VEGF
165
were reported to induce
transient angiogenic effects (increased vessel density and perfusion) in mouse skeletal muscle.
9
These angiogenic effects peaked at four to seven days after injection, and then sharply
decreased, reaching negligible levels at two weeks. The effects of VEGF
165
were slightly more
prolonged. Lymphatic vessels were only very weakly affected by hVEGF-D in this system.
For the current studies we used rAAV-delivered transgene expression, as it was previously
reported to provide more prolonged transgene expression combined with lower
immunogenicity.
17–19,32
Using immunohistochemical analysis we could observe a significant
increase in lymphatic as well as blood vessels at two- and four-week time points, although the
overall magnitude of the response depended on the mouse strain and in particular on the
vascular growth factor used. Both effects were more profound when the activated, short forms
were used, as compared with the full-length forms. Full-length mVEGF-D was not angiogenic
or lymphangiogenic in our assays and full-length mVEGF-C was only able to induce formation
of lymphatic vessels, apparently originating from the preexisting lymphatics found in between
the myofibers.
33
hVEGF-Dsf is significantly less active than hVEGF-Csf at inducing proliferation of VEGFR-2/
BaF3 and VEGFR-3/BaF3 cells. Furthermore, neither mVEGF-Dfl nor mVEGF-Dsf was
active in the VEGFR-2/BaF3 assay, consistent with the previously reported lack of mVEGF-
D/mVEGFR-2 interaction.
21
However, our in vivo data (immunohistochemistry for PECAM-1,
MECA32 and LYVE-1, ultrasound analysis of muscle perfusion, visualization of blood vessels
with DiI and studies of the functionality of newly formed lymphatic vessels by FITC-lectin
uptake and transport) conclusively demonstrate that the angiogenic activity of rAAV-delivered
VEGF-Dsf is comparable to that of VEGF-Csf, although mVEGF-Dsf shows significantly
weaker smooth muscle cell recruiting activity than mVEGF-Csf, at least in the FvB/NJ and
C57Bl/6J mice.
One explanation for the apparent discrepancy between in vitro and in vivo data is that human
or mouse VEGF-Dsf binds to VEGFR-3 in vitro with at least 20-fold lower affinity than VEGF-
Csf.
2,5,21
Thus, to obtain equal stimulatory activity of VEGF-C and VEGF-D on receptor-
bearing BaF3 cells in vitro, the proteins have to be added into the cell cultures in significantly
different quantities, as here demonstrated using the BaF3 cell-based assays. Yet, in vivo, rAAV-
infected myofibers provide continuous expression and supply of the transgenic protein, which
may accumulate in high enough local concentrations to stimulate endothelial cell growth.
In the current study, as well as in previous reports, VEGF-D was shown to induce an angiogenic
response.
9,13,14
Although VEGF-D does not activate mouse VEGFR-2,
21,22
it is possible that
VEGFR-3 is involved in the angiogenic response.
10,34,35
On the other hand, we observed
accumulation of CD45- and F4/80-positive cells, especially in the C57Bl/6J mice, that could
produce angiogenic factors at sites of rAAV transgene expression. The accumulation of bone
marrow-derived inflammatory cells capable of producing a range of biologically active
molecules (including VEGF, TGFβ, PDGF-B, and others), can provide additional angiogenic
and arteriogenic signals at sites of adult angiogenesis.
36–38
Furthermore, inflammatory cells
and especially macrophages can also promote lymphangiogenesis by synthesis and secretion
of VEGF-C and VEGF-D.
39–41
Our functional tests confirmed that the newly generated blood and lymphatic capillaries were
perfused and thus functional. Transgene expression in muscle tissue at four weeks after vector
injection was consistent with morphological and functional changes observed at that time.
However, only some myofibers expressed the transgenes (unpublished data), similar to what
has been reported for rAAV8-LacZ-injected canine muscle, where the initially high numbers
of β-galactosidase positive myofibers were significantly reduced 4 to 8 weeks after injection.
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42
It has been reported, that the majority of stably transduced host cells apparently maintain
recombinant AAV genome as extrachromosomal episomes.
43
The strongest stimulator of BaF3 cell proliferation was the hVEGF-Csf C137A mutant, which
showed improved dimer stability in multiple assays. Evidently, disulfide reducing agents that
lead to full dissociation of the native hVEGF-Csf only partially affected hVEGF-Csf C137A
homodimers. It should be noted that the cysteine residue corresponding to C137 in human
VEGF-C is not conserved outside of the VEGF-C/D subfamily, and according to our structural
model it is located next to the putative intermolecular disulfide bridge. We therefore suggest
that this cysteine residue makes the intermolecular disulfide bond unstable, resulting in non-
covalent homodimers of wild-type VEGF-Csf.
6
Replacing cysteine-137 with alanine may lead
to the stabilization of the intermolecular disulfide bond and ultimately to a more stable hVEGF-
Csf C137A homodimer. As mentioned previously, this more stable dimeric form of VEGF-C
can bind to and activate the VEGFR-3 and VEGFR-2 receptors more efficiently and the
corresponding change in VEGF-D has a similar effect
44
.
Our immunohistological data demonstrate that, compared to hVEGF-Csf, the hVEGF-
CsfC137A mutant shows significantly increased arteriogenenic activity (SMA panel in Figure
4A), but not increased lymphangiogenic activity. In previous studies of adenovirally
transfected mouse and rabbit skeletal muscles, human VEGF-D was also demonstrated to
possess arteriogenic activity.
9,13
The differences between previously published data and our
own might be attributable to differences in target cells and to varying levels of transgene
expression. – Unexpectedly, all C57Bl/6J mice transduced with rAAV8-VEGF
165
died within
10 days, while mice transduced with corresponding doses of rAAV8-CAC survived and
showed strong angiogenesis in the injected muscles. CAC was also a stronger angiogenic
inducer than human VEGF-C or VEGF-D, but it did not promote lymphangiogenesis. Thus
this chimeric factor has interesting properties that might be exploited for potential gene therapy.
In conclusion, we have found that rAAV-delivered activated VEGF-C and VEGF-D and an
engineered VEGF-C variant can all induce blood and lymphatic vessels in mouse skeletal
muscle. The high arteriogenic activity of the hVEGF-Csf C137A growth factor seems to be
attributable to its enhanced dimer stability due to the formation of stable interchain disulfide
bonds. The most active arteriogenic factors also demonstrated a trend towards higher
angiogenic activity, and the newly generated blood and lymphatic vessels were determined to
be functional. Future goals now will include determining the therapeutic benefit of these factors
in conditions of ischemia or muscle edema.
Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
Acknowledgments
We thank Professor Seppo Sarna and Drs. Paul Bromann, Caroline Heckman and Tuomas Tammela for critical
comments on the manuscript; Tapio Tainola, for expert technical assistance; the Biomedicum Molecular Imaging Unit
for microscope support and maintenance and the staff at the Biomedicum Helsinki and the Haartman Institute Animal
Facilities for excellent animal husbandry.
Sources of Funding
This study was supported by NIH grant 5-R01-HL075183-02; the Finnish Technology Development Centre, the
Academy of Finland Council of Health, the Sigrid Juselius Foundation, the Finnish and European Foundations for the
Study of Diabetes and the Novo Nordisk Foundation.
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Figure 1. Biochemical characterisation of recombinant growth factors
A, Alignment of the human (h) and mouse (m) VEGF-Csf (Csf) sequences. The amino acid
residue differences (V146/A142, S179/G175) are indicated in blue and green and the mutated
residue (C137) in red. B, Soluble VEGFR-2 and VEGFR-3 precipitation of human and mouse
factors followed by SDS-PAGE analysis under reducing conditions. Empty plasmid was used
as the mock control. C, Coprecipitation of mouse full-length (VEGF-Cfl) and VEGF-Dfl with
VEGFR-2 or VEGFR-3. D, MTT cell survival assay with VEGFR-2/BaF3 cells for the VEGF-
Csf native and mutant molecules produced in transfected 293T cells. Mouse and human VEGF-
Csf are denoted by mCsf and hCsf, respectively. V146A, S179G and C137A mutants of
hVEGF-Csf are denoted as hCsf146A, hCsf179G, and hCsf137A, respectively. E, The same
assay as in D, but utilizing VEGFR-3/BaF3 cells. In D and E, statistically significant (P<0.05)
differences between the activities of certain proteins over range of studied concentrations are
indicated by square brackets.
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Figure 2. Biochemical properties of wild-type versus mutant VEGF-Csf and VEGF-Dsf
A, A 3-D computer model (SWISS-MODEL
45
) shows the putative location of the C137A
mutation in the VEGF-Csf structure. The mutant residue (shown by red in Figure 1A) is marked
with arrows. The two antiparallel polypeptide chains of hVEGF-Csf homodimer are shown in
grey and green. The hVEGF-Csf model is based on the known crystal structure of VEGF amino
terminal residues 8-109
23
and other VEGF family proteins
24,25
. B, Analysis of the VEGFR-3
binding affinities of human VEGF-Csf and its C137A mutant by isothermal titration
calorimetry. The proteins were produced as described in Materials and Methods. C, ELISA-
based competitive binding assay with VEGF-Csf and its C137A mutant. D, VEGFR-3 co-
precipitation of [
35
S]-labelled human VEGF-Csf mutants or wild type proteins, which were
obtained from the media of 293T cells transfected with the corresponding plasmids (Di - dimer,
Mo - monomer). Mock represents the supernatant of 293T cells transfected with empty plasmid.
E, SDS-PAGE of purified VEGF-Csf and its C137A mutant treated with varying
concentrations of DTT. The proteins were stained with Coomassie Blue. Growth factor
abbreviations are described in Figure 1 legend.
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Figure 3. Blood vascular and lymphatic endothelial staining of the target muscles four weeks after
gene transduction
Immunostaining for PECAM-1 (endothelial cells), MECA32 (blood vessel endothelial cells),
LYVE-1 (lymphatic endothelial cells), SMA (smooth muscle cells and pericytes), and CD45
(leukocytes) frozen sections of the t.a. muscles of FVB/NJ mice. Full-length forms of mouse
VEGF-Cfl and VEGF-Dfl were tested along with mouse and human VEGF-Csf and VEGF-
Dsf. A, Quantification of the immunostaining was perfomed as described in Materials and
Methods. * marks statistical significance at P<0.05, when compared to the HSA control. Square
brackets with # mark statistical significance at P<0.05 between the indicated experimental
groups. B, Representative images of mouse factor immunostaining are shown. The
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corresponding PECAM-1/SMA immunostaining patterns are shown in Online Figure III. Note
that only mVEGF-Csf and VEGF
165
induced significant recruitment of smooth muscle cells.
Scale bars here and in all other figures are 100 μm, unless otherwise indicated.
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Figure 4. Blood vascular, lymphatic endothelial, and smooth muscle staining two weeks after gene
transduction
Double-immunostaining of the indicated antigens in frozen muscle sections from C57Bl/6J
mice. A, Quantification of the immunostaining was performed as in Fig. 3. B, Representative
images of hVEGF-Csf C137A-, CAC-, and HSA-treated muscle samples. Growth factor
abbreviations are described in the Figure 1 legend. Note that while hVEGF-Csf C137A induces
growth of both blood (MECA32-positive) and lymphatic (LYVE-1-positive) vessels, CAC
induces only blood vessels. It should be noted that some of the inflammatory cells were also
PECAM-1 positive.
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Figure 5. Muscle perfusion two weeks after transduction of rAAV vectors
C57Bl/6J mice were separated into 7 groups, and 4 animals per group were injected with vectors
encoding the indicated growth factors. Blood flow in t.a. muscle was quantified by Doppler
ultrasound. Significance values were determined between the test groups and the negative
control group (HSA). The bars indicate ± s.e.m.; * marks statistically significant differences
to HSA control. # marks statistical significance between CAC and any other experimental
group. Representative 2-D images from the scanning of the muscles transduced with the various
vectors are indicated.
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Figure 6. Comparison of perfusion staining of vessels induced by VEGF-C/-D, CAC and VEGF
Intramuscular blood vessels were stained by perfusion with the lipophilic dye DiI two weeks
after rAAV transduction. Note that both VEGF-C and VEGF-D increase vessel density and
size. In CAC treated muscles, vessel density is markedly increased and vessel morphology
most similar to that of muscle injected with the control vector (HSA), whereas VEGF induced
the formation of angioma-like structures (arrowheads). Capillary-sized vessels are indicated
by arrows.
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Figure 7. Mouse VEGF-Csf and VEGF-Dsf induce formation of functional lymphatic vessels
A, Lymphatic dye uptake and transfer were imaged 45 minutes after FITC-lectin injection into
the distal (lower) part of t.a. muscles four weeks post-transduction with rAAVs encoding the
indicated proteins. The stained sections were prepared from the regions indicated by the
punctuated lines. Arrows indicate sites of FITC-lectin injection. The scale bar indicates 1 mm.
B, FITC-lectin, nuclear (Hoechst 33258), and anti-LYVE-1 immunostaining were performed
using muscle sections obtained from the muscle areas indicated in A.
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