Dual effects of VEGF-B on activating cardiomyocytes and cardiac stem cells to protect the heart against short- and long-term ischemia–reperfusion injury
Li et al. J Transl Med
Dual effects of VEGF-B on activating cardiomyocytes and cardiac stem cells to protect the heart against short- and long-term ischemia-reperfusion injury
Guo‑hua Li 1 2
Bin Luo 2
Yan‑xia Lv 2
Fei Zheng 0
Lu Wang 0
Meng‑xi Wei 0
Xiany‑u Li 4
Lei Zhang 0
Jia‑ning Wang 0
Shiy‑ou Chen 3
Jun‑Ming Tang 0 2
Xiaohua He 1
0 Department of Cardiology and Institute of Clinical Medicine, Renmin Hospital, Hubei University of Medicine , Shiyan, Hubei Province 442000 , China
1 School of Basic Medical Sciences, Wuhan University , Wuhan 430071, Hubei Province , China
2 Department of Physiology, School of Basic Medical Sciences, Hubei University of Medicine , Shiyan, Hubei Province 442000 , China
3 Department of Physiol‐ ogy and Pharmacology, University of Georgia , Athens, GA 30602 , USA
4 Depart‐ ment of Pathophysiology, School of Basic Medical Sciences, Hubei University of Medicine , Shiyan, Hubei Province 442000 , China
Aims: To investigate whether vascular endothelial growth factor B (VEGF‑ B) improves myocardial survival and cardiac stem cell (CSC) function in the ischemia-reperfusion (I/R) heart and promotes CSC mobilization and angiogenesis. Methods and results: One hour after myocardial ischemia and infarction, rats were treated with recombinant human VEGF‑ B protein following 24 h or 7 days of myocardial reperfusion. Twenty‑ four hours after myocardial I/R, VEGF‑ B increased pAkt and Bcl‑ 2 levels, reduced p‑ p38MAPK, LC3‑ II/I, beclin‑ 1, CK, CK‑ MB and cTnt levels, triggered cardiomyocyte protection against I/R‑ induced autophagy and apoptosis, and contributed to the decrease of infarction size and the improvement of heart function during I/R. Simultaneously, an in vitro hypoxia‑ reoxygenation (H/R)‑ induced H9c2 cardiomyocyte injury model was used to mimic I/R injury model in vivo; in this model, VEGF‑ B decreased LDH release, blocked H/R‑ induced apoptosis by inhibiting cell autophagy, and these special effects could be abolished by the autophagy inducer, rapamycin. Mechanistically, VEGF‑ B markedly activated the Akt signaling pathway while slightly inhibiting p38MAPK, leading to the blockade of cell autophagy and thus protecting cardiomyocyte from H/R‑ induced activation of the intrinsic apoptotic pathway. Seven days after I/R, VEGF‑ B induced the expression of SDF‑ 1α and HGF, resulting in the massive mobilization and homing of c‑ Kit positive cells, triggering further angiogenesis and vasculogenesis in the infracted heart and contributing to the improvement of I/R heart function. Conclusion: VEGF‑ B could contribute to a favorable short‑ and long‑ term prognosis for I/R via the dual manipulation of cardiomyocytes and CSCs.
Cardiac stem cells; VEGF‑ B; Mobilization; Apoptosis; Angiogenesis
Ischemic heart disease (IHD) is the leading cause of death
in the modern world. Myocardial infarction (MI) is
usually initiated by myocardial ischemia resulting from the
narrowing of coronary arteries due to atherosclerosis. At
the clinical level, a number of cardiovascular intervention
techniques, such as percutaneous coronary intervention
(PCI) and cardiac bypass surgery, have been developed to
restore myocardial perfusion [
]. Reperfusion occurs as
a result of these interventions (particularly PCI); in short
term, intervention should overcome ischemia–reperfusion
injury (I/R), while in the long term, vascular restenosis
should be effectively prevented via the activation of
angiogenesis and vasculogenesis. However, the most effective
therapeutic modality has not been clearly established.
The VEGF family of archetypal angiogenic growth
factors consists of five secreted dimeric glycoprotein
growth factors in mammals: VEGF (or VEGF-A),
VEGFB, VEGF-C, VEGF-D and PlGF (placenta growth
]. Although VEGF-mediated angiogenesis plays a
critical role in the repair of ischemia/infarction, which is
characterized by reduced blood supply to the heart [
until recently, VEGF-B was an exception to the family’s
classical role in promoting angiogenesis [
levels are highest in high metabolic tissues, such as the
myocardium, skeletal muscle and adipose tissue [
The absence of VEGF-B has been reported to lead to the
decreased expression of fatty acid (FA) transport proteins
in endothelial cells, which is associated with reduced
lipid droplets in skeletal muscle and cardiomyocytes and
improved insulin resistance in diabetes models [
VEGF-B is markedly down-regulated in the infarcted
]. The over-expression of VEGF-B improved
cardiac contractility in rats after experimental MI .
However, it is unknown whether VEGF-B plays a role
in short- and long-term prognosis following myocardial
ischemia for the development of heart failure.
In our previous study, cardiac stem cells (CSC), which
are characterized by their migratory capabilities and
potential for differentiating into cardiomyocytes and
vascular cells, showed traits of VEGFR1 expression, were
partly involved in VEGF-A-induced CSC migration, and
contributed to the repair of the infarcted heart by
inducing angiogenesis [
]. In addition to protecting
cardiomyocytes, VEGF-B strongly induced myocardium-specific
angiogenesis and arteriogenesis through VEGFR1 [
] and partly contributed to the involvement of
endothelial cells in angiogenesis [
]. Of interest, CSCs are
directly involved in the formation of the large coronary
]; thus, they predict the evolution of ischemic
cardiomyopathy following revascularization [
present, the relationship between VEGF-B-induced
angiogenesis and CSCs during the process of myocardial
ischemia or infarction, particularly in long-term I/R, is
unknown. Angiogenic cytokine therapy has been widely
regarded as an attractive approach for treating ischemic
heart disease and for preventing vascular restenosis
following cardiovascular intervention [
1, 2, 4
we hypothesize that VEGF-B plays a pivotal role in the
short- and long-term prognosis of myocardial I/R via the
dual manipulation of cardiomyocytes and CSCs.
All animal experiments were performed according to
the Guide for the Care and Use of Laboratory Animals
recommended by the US National Institutes of Health.
All protocols for animal studies were permitted by the
Institutional Animal Care and Use Committee of Hubei
University of Medicine.
Heart ischemia–reperfusion injury model
To observe whether VEGF-B protects against myocardial
I/R injury in vivo, a rat model of myocardial I/R injury
was established. Male Sprague–Dawley rats (240–280 g)
were obtained from the Experimental Animal Center at
Hubei University of Medicine and housed at an
appropriate temperature (25 °C) with relative humidity (55 %),
a fixed 12-h light/dark cycle and free access to food and
water. The animals were randomly divided into four
groups, as follows: a sham-operated group, an I/R injury
group (I/R), a VEGF-B (1.0 μg/kg) group and a
VEGFB (10 μg/kg) group. The in vivo doses of VEGF-B were
selected according to a previous study [
solution 200–300 μL (1.0 or 10 μg/mL) was injected with
a 30-gauge tuberculin syringe into four sites
(approximately 50–75 μL per site) into each I/R heart; volumes
were determined according to the rat’s body weight. Two
injection sites were in the myocardium bordering the
ischemic area, and two were within the ischemic area.
The animals were anesthetized with ketamine (50 mg/
kg, ip) and xylazine (10 mg/kg, ip) and ventilated during
left anterior descending coronary artery (LAD) ligation
using a Colombus ventilator (HX-300, Taimeng
Instruments, China). Surgery was performed under sterile
conditions. The LAD was ligated for 1 h, and then opened for
treatment with VEGF-B (local injection of the left
myocardium, four sites at 50 μL per site) for 24 h or 7 days
of reperfusion. In the sham-operation group, the rats
underwent identical surgery but without ligation of the
coronary artery. Buprenorphine hydrochloride (0.05 mg/
kg, sc) was administered one time after the procedure.
Measurement of creatine kinase (CK), CK‑MB activity and cardiac troponin T (cTnT)
This procedure was described in detail elsewhere [
Briefly, 24 h after treatment, blood samples were
centrifuged at 3500 rpm for 15 min at 4 °C; then the serum
was collected. Subsequently, according to a handbook
of experimental operations, CK activity (JianCheng
Bioengineering Institute, Nanjing, China), CK-MB activity
(Rapidbio, USA) and cardiac troponin T (cTnT)
(Rapidbio, USA) levels, as enzymatic diagnostic indexes of
myocardial injury, were detected and analyzed.
Hemodynamic measurement was performed as described
]. Briefly, after 24 h of reperfusion, the
animals were anesthetized with ketamine (50 mg/kg, ip) and
xylazine (10 mg/kg, ip), and the left carotid artery was
exposed. A catheter filled with heparinized (10 U/ml)
saline solution was connected to a pressure transducer
(Chengdu Taimeng Technology Co., Ltd., China) and
then advanced into the left ventricle to record
ventricular pressure for 15 min. Hemodynamic parameters were
monitored simultaneously and recorded using Biological
signal acquisition system BL-420S (Chengdu Taimeng
Technology Co., Ltd., China).
Twenty-four hours after reperfusion, the hearts were
removed and washed with K-H buffer at room
temperature for 3 min, frozen at −20 °C for 1 h and
transverse-sectioned into five parts (thickness, 2–5 mm).
The sections were then incubated in 1 % 2, 3,
5-triphenyltetrazolium chloride (TTC, Sigma, USA) at 37 °C for
15 min. The infarcted myocardium was not stained by
the TTC and appeared white in color; meanwhile, the
non-ischemic myocardium was stained by the TTC and
appeared brick-red in color. The infarction size was
calculated by multiplying the planimetered areas by the slice
thickness. The infarction size was expressed as the
percentage of the left ventricular size of each heart.
Cardiomyocyte apoptosis assay in vivo
To analyze cardiomyocyte apoptosis, 24 h after
reperfusion, the hearts were removed, fixed in 4 %
paraformaldehyde and embedded in an optimum cutting temperature
compound (Fisher Scientific). Serial transverse Sections
(5 μm) were cut across the longitudinal axis of the heart
and mounted on slides. After a brief washing in
phosphate-buffered saline (PBS), the heart sections were
incubated in a blocking buffer [PBS containing 1 % fetal
calf serum (FCS) and 0.1 % Triton X-100] at room
temperature for 1 h. Cardiomyocyte apoptosis was detected
using the methods described in the manual of the
InSitu Apoptosis Detection Kit (MM_NF-S7165#,
Millipore, UEA). Five fields from each section (n = 3) were
randomly selected. The number of apoptotic cells was
counted manually by two pathologists, who were
unaware of the experimental design. The rate of
apoptosis was calculated as the percentage of all cells per high
power field (HPF; 400×).
Seven days after I/R, the heart tissues were fixed in 4 %
paraformaldehyde and embedded in optimum cutting
temperature compound (Fisher Scientific). Serial
transverse Sections (5 μm) were cut across the longitudinal
axis of the heart and washed with phosphate-buffered
saline (PBS). The heart sections were then incubated in
a blocking buffer at room temperature for 1 h. The
sections were incubated in antibodies (diluted 1:250 in
blocking buffer) at 4 °C overnight for primary antibodies
and at room temperature for 2 h for secondary
antibodies. The primary antibodies used were mouse anti-rat
vWF VIII, rabbit anti-rat a-SMA (Santa Cruz) and
antirat c-kit (Abcam). The secondary antibodies were
FITCconjugated anti-rabbit IgG, FITC-conjugated anti-rat
IgG, and FITC-conjugated anti-mouse IgG (Santa Cruz)
]. Images of c-Kit positive cells or vessels were acquired
under microscopy (magnification, 400×) for five random
fields in the infarcted area of each transverse slice for all
three groups. The number of c-Kit positive cells or
vessels in the infarcted areas was counted manually by two
pathologists who were unaware of the experimental
design and are presented as the mean of c-Kit positive
cells or blood vessels per unit area (0.2 mm2).
Culture of H9c2 rat cardiomyocytes
The rat embryonic ventricular myocardial cell line H9c2
was purchased from the Cell Bank of the Chinese
Academy of Sciences (Shanghai, China). H9c2 cells were
cultured as described previously [
Hypoxia‑reoxygenation of H9c2 Cells
The in vitro hypoxia-reoxygenation of H9c2 cells
occurred as described previously [
]. In brief, H9c2
cells were treated with or without VEGF-B (20 ng/ml,
Peprotech, USA) in EBSS after H9c2 cells culture under
low-oxygen conditions (95 % N2 + 5 % CO2) for 6 h in
a humidified hypoxia chamber (Stem Cell Technology,
USA). The in vitro doses of VEGF-B (0.1, 1, 10, 20 and
50 ng/ml) were determined as described in detail
]. After hypoxic incubation, the medium was
replaced with 15 % FBS medium under normoxic
conditions (95 % air + 5 % CO2) for reoxygenation for 3 h.
Cell apoptosis assay in vitro
Cell apoptosis was determined as described previously
]. According to the cell apoptosis protocol, H9c2 cells
were resuspended in 500 µl binding buffer after washing
and then incubated with Annexin V solution for 3 min,
followed by PI solution (Bender MedSystems, Austria)
for 15 min. The apoptotic cells were analyzed with flow
Lactate dehydrogenase (LDH) release
The LDH level is an indicator of cellular injury [
The LDH release was measured using commercial kits
(JianCheng Bioengineering Institute, China).
Cell autophagy assay
H9c2 cells were cultured on a chamber slide to 50–60 %
confluence. The cells were transfected with
Ad-mRFPGFP-LC3-I/II (MOI: 100, Hanbio, Co, China) for 48 h
and then treated with VEGF-B (10 ng/ml) according the
protocol for the hypoxia-reoxygenation of H9c2 Cells.
The H9c2 cells or heart tissues were lysed in RIPA
buffer, and 30 µg of proteins were separated in a 12 %
SDS–PAGE gel and transferred onto a nitrocellulose
membrane (Millipore). The membrane was rinsed with
Tris-buffered saline (TBS) containing 0.1 %
Tween20 (TBST) and blocked with 5 % fat free milk in TBS
at room temperature for 1 h. The membrane was then
incubated with anti-Akt, anti-phospho-Akt, anti-Bcl-2,
anti-Bax, anti-beclin1, anti-p38MAPK, anti-ERK1/2,
anti-pp38MAPK, anti-pERK1/2 (Cell Signaling
Technology, 1:500), anti-SDF-1α, anti-HGF (Santa Cruz) or
antiα-Tubulin antibody (Sigma, 1:5000) and then incubated
with horseradish peroxidase-conjugated secondary
antibodies (1:10,000 dilution; Santa Cruz). The immunoblots
were detected using enhanced chemiluminescence
reaction (Amersham Pharmacia Biotech) and measured with
All data are expressed as mean ± SD. The parameters
of two groups were compared using the unpaired t test.
The parameters of more than two groups were compared
using one-way analysis of variance. P values <0.05 were
considered statistically significant.
VEGF‑B reduced infarct size and improved heart function
To observe the effect of VEGF-B on heart I/R injury
in vivo, we performed I/R experiments that mimic
therapeutic reperfusion in human patients. Rat hearts
were subjected to ischemia by ligating the LAD for 1 h,
followed by reperfusion for 24 h to assess the effect of
VEGF-B on myocardial infarct size and heart function
in an I/R model in vivo. One-percent TTC staining was
used to evaluate the infarct size of rat hearts exposed
to reperfusion with or without VEGF-B treatment. The
results showed that 1.0 and 10 μg/kg VEGF-B treatment
significantly reduced the infarct size in vivo compared
with the I/R group. Furthermore, the 10 μg/kg
VEGFB group showed a greater decrease in the infarct size
compared with the 1.0 μg/kg VEGF-B group (Fig. 1a, b).
In line with the change in infarct size, the left ventricle
function of the heart in both VEGF-B groups was
significantly improved compared with the I/R group.
Furthermore, the higher dose of VEGF-B had a much greater
effect on increasing LVSP and ±dp/dtmax and
decreasing LVEDP compared with the lower dose of VEGF-B
(Fig. 1c–f ).
VEGF‑B suppressed CK, CK‑MB activity, and cTnT levels on heart I/R injury
To monitor myocardial damage, CK, CK-MB, and cTnT
activity was detected to evaluate myocardial injury. The
results showed that CK, CK-MB, and cTnT activity were
obviously increased in the I/R group compared with the
sham group (Additional file 1: Figure S1A–D).
Interestingly, compared with the I/R group, the VEGF-B groups
all had significantly reduced CK, CK-MB activity and
cTnT levels. Furthermore, high-dose VEGF-B had
better protective effects against heart I/R (Additional file 1:
Figure S1A–C). These data suggest that VEGF-B inhibits
I/R-mediated myocardial injury.
VEGF‑B inhibits I/R‑mediated cardiocyte apoptosis in vivo
To further assess the potential protective mechanism of
VEGF-B in heart I/R injury, cardiomyocyte apoptosis, a
classical marker of myocardial I/R damage, was examined
after VEGF-B treatment. We found that VEGF-B
significantly decreased the apoptosis rate of cardiocytes in
the in vivo I/R model (Fig. 2a, b). Bcl-2/Bax, two typical
indicators of cardiocyte apoptosis, were used to
evaluate the effect of VEGF-B on myocardium exposed to I/R.
The results showed that Bcl-2 expression was obviously
induced and Bax expression was significantly decreased
in VEGF-B-treated hearts compared with the I/R group
(P < 0.05). Finally, Bcl-2/Bax regulation was generally
involved in multiple signaling pathways, such as PI3K/
Akt, ERK1/2 and p38MAPK, et al. [
]. The changes in
nonphosphorylated and phosphorylated Akt, ERK1/2
and p38MAPK were analyzed and compared with
those of the I/R group. Akt, ERK1/2 and p38MAPK and
pERK1/2 levels were not markedly increased
(Additional file 1: Figure S2), but pAkt levels were obviously
increased in both the VEGF-B (1.0) and VEGF-B (10)
group (Fig. 2c–e). Meanwhile, p-p38MAPK levels were
decreased in the two VEGF-B groups. Furthermore, the
higher dose of VEGF-B had better benefits for increasing
pAkt levels and decreasing p-ERK1/2 and p-p38MAPK
In addition, because VEGF-B is a newly identified
metabolic factor [
] associated with cell autophagy, we also
examined changes in LC3-II/I, an indicator of autophagy.
We found that the LC3-II/I rates were increased in I/R
group compared to with the sham group and that
compared with the I/R group, the two VEGF-B treatment
groups showed decreased LC3-II/I rates, and the higher
dose of VEGF-B had a better effect on reducing LC3- II/I
rates (Fig. 2c, i).
These data suggest that VEGF-B could prevent
I/Rmediated cardiocyte autophagy and apoptosis associated
with the PI3K/Akt and p38MAPK signaling pathways.
VEGF‑B reversed the changes in H/R‑induced H9c2 cell apoptosis in vitro
To develop a detailed description of the role and
mechanism of VEGF-B in I/R injury, an in vitro H/R-induced
H9c2 cell damage model was established to mimic the
in vivo myocardial I/R model. DAPI staining showed that
H/R injury caused significant apoptosis in H9c2 cells, as
shown by condensed nuclei, an indicator of apoptosis.
However, VEGF-B restored H9c2 nuclei to their normal
morphology (Fig. 3a). The appearance of
phosphatidylserine (PS) in the outer leaflet of the phospholipid bilayer
without a disruption in membrane integrity is one of
the earliest characteristics of apoptotic cells [
cytometry assay was used to evaluate the changes in cell
apoptosis using Annexin V/PI staining, which indicates
PS production. As Fig. 4b shows, I/R induced H9c2 cell
apoptosis. However, VEGF-B attenuated the number
of Annexin V/PI-positive cells, suggesting that VEGF-B
inhibited H/R-induced apoptosis (Fig. 3b). Furthermore,
the protective effects of VEGF-B on H9c2 cells exposed
to H/R showed dose-dependent characteristics (Fig. 3c).
LDH release is an indicator of cellular injury [
Compared with untreated cells, LDH levels were
markedly increased by H/R injury. VEGF-B treatment,
however, decreased H/R-induced LDH release (Fig. 3d).
Meanwhile, H/R obviously induced Bax expression and
decreased Bcl-2 expression in H9c2 cells, and VEGF-B
treatment significantly increased Bcl-2 expression while
reducing Bax expression in H/R-injured H9c2 cells in a
dose-dependent manner (Fig. 3e–h). These data
suggest that VEGF-B is able to protect cardiomyocytes from
H/R-induced injury in a dose-dependent manner.
VEGF‑B regulated cell apoptosis in vitro
A recent publication indicates that apoptosis is
mediated by numerous signaling pathways, such as PI3K/Akt,
ERK/1/2 and p38 MAPK [
]. To test whether these
mechanisms apply to VEGF-B-induced myocardium protection,
we used VEGF-B to stimulate cardiomyocytes before H/R
injury. We found that VEGF-B obviously enhanced pAkt,
slightly increased pERK1/2, and decreased p-p38MAPK
without affecting their expression (Additional file 1:
Figure S3) in a dose-dependent manner (Fig. 4a–d). To
confirm whether these signaling molecules were involved
in VEGF-B-mediated anti-apoptosis, special inhibitors
were used to assess the relationship between them. The
results showed that the ERK1/2 inhibitor PD98059 did
not obviously abolish the anti-apoptotic effect of VEGF-B,
but the p38MPAK inhibitor SB203580 and the PI3K
specific inhibitor LY294002 obviously blocked it (Fig. 4e, f ),
suggesting that VEGF-B prevents apoptosis in
cardiomyocytes through PI3K/Akt and p38MAPK signaling.
VEGF‑B inhibited H/R‑induced H9c2 cell apoptosis through the regulation of Bcl‑2/Bax expression mediated by PI3K/Akt signaling
Because the cell survival regulators Bcl-2 and Bax are
regulated by PI3K/Akt signaling [
] and VEGF-B had a
regulatory effect on Bcl-2/Bax expression (Fig. 3e–h), we
examined whether VEGF-B-mediated Bcl-2/Bax
regulation was involved in PI3K/Akt signaling. As shown
in Additional file 1: Figure S4A, VEGF-B markedly
increased Bcl-2 while decreasing Bax expression,
suggesting that VEGF-B prevents apoptosis in H9c2 cells
by readjusting the expression and ratio of Bcl-2 and Bax
(Additional file 1: Figure S4A). LY294002 blocked
VEGFB-mediated Bcl-2 and Bax expression (Additional file 1:
Figure S4A, 4D–4F), suggesting that VEGF-B regulates
Bcl-2/Bax expression through the PI3K/Akt signaling
VEGF‑B inhibited I/R‑induced autophagy in a dose‑dependent manner
VEGF-B is a newly identified metabolic factor [
that could be associated with cell autophagy. To
determine whether VEGF-B affected autophagy, as shown
in Fig. 5a, H/R increased the expression of
autophagyrelated proteins in H9c2 cells. VEGF-B reduced the
levels of autophagy-related proteins and the ratio of
LC3-II/I seen on western blot analysis (Fig. 5a, b). The
GFP-RFP-LC3 adenovirus (Ad-mRFP-GFP-LC3) system
was further used to confirm the induction of autophagy
using punctate forms, which represent autophagosome
]. As Fig. 3c shows, after infection with
Ad-mRFP-GFP-LC3, we observed the successful
introduction of this adenovirus and both fluorescent
proteins. In addition to accumulation of LC3, there were
more red puncta in the H/R-induced H9c2 cells than
in the control cells. Furthermore, VEGF-B markedly
decreased the number of red puncta in a
dose-dependent manner. These results further confirmed the
induction of autolysosome formations in the H/R model,
indicating that VEGF-B mediated an anti-autophagy
flux in H9c2 cells.
VEGF‑B inhibited H9c2 cell apoptosis by inhibiting
To further explore the involvement of VEGF-B in H9c2
cell apoptosis in the H/R-induced autophagy model,
the autophagy inducer rapamycin and the autophagy
inhibitor chloroquine were used to determine whether
VEGF-B’s effect on autophagy involved H9c2 cell
apoptosis. As Fig. 6a and c show, rapamycin reversed the
VEGF-B-mediated anti-apoptosis effects, the
VEGF-Bmediated anti-autophagy effects and the LC3-II/I rates
in the H/R model (Fig. 6d, e), suggesting that
VEGFB-mediated anti-autophagy plays a critical role in the
protective effects of VEGF-B in H9c2 cells under H/R
conditions. Additionally, chloroquine enhanced the
VEGF-B-mediated anti-autophagy and anti-apoptosis
effects in H/R-induced cell injury. These data
demonstrate that VEGF-B could inhibit H/R-induced cell
apoptosis by blocking autophagy.
VEGF‑B inhibited autophagy‑mediated H9c2 cell apoptosis
Because the p38MAPK and PI3K/Akt signaling pathways
play important roles in both apoptosis and autophagy [
], the PI3K-specific inhibitor LY294002, an autophagy
inducer, was used to observe the effects of VEGF-B on
H9c2 cells under H/R conditions . As Fig. 7a, b show,
the expression of autophagy-related proteins and
LC3II/I rates increased and the autophagy flux (shown in
red) was enhanced in H/R-induced injury of H9c2 cells.
VEGF-B reduced the levels of autophagy-related proteins
and LC3-II/I rates, and the special effects of VEGF-B on
autophagy could be abolished by the PI3K-specific
inhibitor LY294002, suggesting that VEGF-B inhibited autophagy
via PI3K/Akt signaling (Fig. 7a–d). Interestingly, the special
effects of VEGF-B on anti-autophagy could not be abolished
by the ERK1/2 inhibitor PD98059 but could be abolished by
the p38MPAK inhibitor SB203580 (Additional file 1: Figure
S5), which was consistent with the finding that PD98059
and not SB203580 inversed VEGF-B-mediated
anti-apoptosis (Fig. 5e). These results imply that VEGF-B can inhibit
autophagy-mediated H9c2 cell apoptosis by regulating the
PI3K/Akt, ERK1/2 and p38MPAK signaling pathways.
VEGF‑B promoted CSC mobilization, angiogenesis and improved heart function
To determine whether VEGF-B can stimulate the
mobilization of CSC in infarcted hearts, we locally injected
VEGF-B into the heart prior to LAD ligation, isolated
the infarcted myocardium 7 days after I/R, and detected
the presence of CSC by immunostaining stem cell
markers for c-kit. A large number of c-kit-positive cells were
observed in the VEGF-B-treated myocardium (Fig. 8a, b).
Because SDF-1α and HGF play important roles in CSC
mobilization in the infarcted myocardium, we detected
SDF-1α and HGF expression in the infarcted heart using
western blotting and found that SDF-1α and HGF
expression obviously increased in infarcted myocardium treated
with VEGF-B in a dose-dependent manner (Fig. 8c–e).
CSCs are closely related to the differentiation of
vascular cells, such as endothelial cells and vascular smooth
muscle cells [
]. Consequently, 7 days after VEGF-B
treatment, angiogenesis in the I/R heart was evaluated
according to microvessel density and artery vessel
density, as indicated by the expression of vWF VIII, a mature
endothelial cell marker, and α-SMA, a vascular smooth
muscle cell marker, respectively (Fig. 8f ). We found that
VEGF-B significantly induced blood vessel formation
in dose-dependent manner, resulting in increased
vessel density (Fig. 8g). An in vitro tube formation assay of
SDF-1α receptor/CXCR4 inhibitor AMD3100 and the
HGF receptor c-Met inhibitor SU11274 were used to
further confirm whether VEGF-B-induced
angiogenesis involved SDF-1α and HGF (Additional file 2). As
shown in Additional file 1: Figure S6, CM-VB induced the
formation of new tubes from c-kit-positive cells in vitro,
and the effect could be partly abolished by AMD3100 or
SU11274. These data demonstrate that VEGF-B induced
angiogenesis mediated by c-kit-positive cells in infarcted
myocardium by activating SDF-1α and HGF signaling.
Furthermore, VEGF-B treatment significantly
improved the function of the left ventricle of the heart,
and the higher dose of VEGF-B had a much greater
effect on increasing LVSP and ±dp/dtmax and decreasing
LVEDP compared with the lower dose (Additional file 1:
In this study, we demonstrate for the first time that
human VEGF-B protein promotes cardiomyocyte
survival by activating the PI3K/Akt/Bcl-2/Beclin1 signaling
pathway after short-term I/R and activates CSC
mobilization and angiogenesis via the induced expression of
SDF-1 and HGF in infarcted heart after long-term I/R.
We also demonstrated improvement in cardiac function
following VEGF-B treatment.
Previous studies demonstrated that VEGF-B had a
protective role in the heart after experimental M) or
angiotensin II-induced heart failure in rats [
The transgene (TG), adenoviral-mediated (Ad) or
adenoassociated virus (AAV) expression of VEGF-B used in
the animal model of MI or heart failure is strong and
long-term, and the safety and controllability of these
vectors are controversial for practical application [
Clinically, human VEGF-B protein should be more far more
acceptable to patients. Recombinant human VEGF-B
protein were therefore used in the present study to
determine the beneficial effects of VEGF-B on cardiomyocyte
protection, CSC mobilization, angiogenesis and cardiac
function of I/R rats.
A recent study shows that VEGF-B induces
compensatory hypertrophy and preserves cardiac function through
VEGFR-1 activation of cardiomyocytes after MI [
Pathways downstream of VEGF-B/VEGFR1, including
the Akt/mTORC1 and ERK1/2, p38MAPK pathways,
which are known to be associated with cardiomyocyte
growth and arteriogenesis, were activated in the heart
31, 33, 34
]. Consequently, VEGF-B’s protective effect
on cardiomyocytes is dependent on PI3K/Akt signaling,
which is in line with the pro-survival function of
]. The anti-apoptotic effects of VEGF-B may be
interpreted using the PI3K inhibitor LY294002 and the
ERK1/2 inhibitor PD98059 to abolish its roles. The
unexpected outcome is that the p38MAPK blocker SB203580
inversed the anti-apoptotic effects of VEGF-B on
H/Rinduced H9c2 cells, while VEGF-B decreased the levels
of p-p38MAPK induced by H/R, indicating that the levels
of p-p38MAPK were related to apoptosis regulation in
VEGF-B-mediated cardiomyocyte protection.
Notably, several studies have suggested that p38 MAPKs
regulated distinct phases of autophagy. p38MAPK can
elicit autophagy via Beclin1 [
Bcl2, a downstream regulator of PI3K/Akt, can inhibit
Beclin1-dependent autophagy, and Beclin1-mediated
autophagy activation during reperfusion is associated
with Bcl-2 down-regulation [
]. Notably, Bcl-2 protein
is also involved in the regulation of apoptosis. Decreased
expression of Bcl-2 may contribute to apoptotic cell death
]. In the present study, VEGF-B increased Bcl-2 protein
levels and inhibited H9c2 cell apoptosis and autophagy
while decreasing the levels of p-p38 MAPKs and Beclin1.
Additionally, the specific effects could be abolished by the
PI3K-Akt inhibitor LY294002 and the p38 MAPK
inhibitor SB203580, demonstrating that VEGF-B prevented
the detrimental effects of autophagy and apoptosis by
controlling the balance between PI3K-Akt and p38 MAPK
The PI3K/Akt pathway is also linked to increased
cardiomyocyte proliferation [
], and the over-expression
of VEGF-B could directly induce the proliferation of
resident c-kit-positive CSC in an angiotensin II-induced heart
failure model [
]. In our study, we found that VEGF-B
increased the number of c-kit-positive CSCs, which are
associated with the activation of the PI3K/Akt pathway, in
the I/R heart. In addition, MSC releases paracrine factors
such as VEGF, SDF1α, and HGF. As a functional
consequence, a conditioned medium of MSCs has a migratory
effect on cardiac resident c-kit-positive CSCs [
which express c-Kit, MDR1, and/or Sca-1 markers, are
self-renewing, clonogenic and multipotent in vitro and
differentiate into cardiomyocytes, smooth muscle cells,
and endothelial cells in vivo. A recent study showed that
endogenous c-kit-positive cells produce few new
cardiomyocytes and mainly generate cardiac endothelial cells at
functionally significant levels within the heart during heart
development, ischemia and infarction states, suggesting
that c-kit-positive cells contribute to heart repair through
enhanced cardiac angiogenesis [
]. Our previous study
showed that VEGF-A directly and indirectly promoted
CSC mobilization in the infarcted heart via the SDF-1α/
CXCR4 axis [
]. In the present study, VEGF-B protein
injection clearly increased the number of resident
c-kitpositive CSCs in the I/R heart while inducing the
expression of SDF-1α and HGF, triggering CSC mobilization,
and inducing angiogenesis and vasculogenesis, leading
to improved cardiac function in the I/R heart. Therefore,
VEGF-B could repair the injured heart by activating the
angiogenesis mediated by CSCs after long-term I/R.
Taken together, the results of the present study suggest
that VEGF-B can improve the short- and long-term
prognosis of I/R via the dual manipulation of cardiomyocyte
protection and CSCs.
Additional file 1. VEGF‑B suppressed CK, CK ‑MB and cTnT in I/R model
of heart in rat in vivo. The levels of total Akt, ERK1 and p38MAPK were not
obviously altered in I/R model of heart in rat in vivo and in H/R model
of H9c2 cells in vitro after VEGF‑B treatment. However, VEGF‑B inhibited
H/R‑induced H9c2 cell apoptosis through up ‑regulating Bcl2 and down‑
regulating Bax expression, these specific effects could be abolished by
PI3K/Akt blocker. And the representative images results showing LC3
changes were shown after SB203580 or PD98059 treatment prior to VEGF‑
B in different groups of H9c2 cells infected with Ad‑ GFP‑RFP ‑LC3. More
importantly, the long‑term effects of VEGF‑B on tube formation of c‑kit
positive CSC in vitro and heart function in vivo were shown.
Additional file 2. Isolated c‑kit+ cells from rat hearts were cultured
and used to evaluate the ability of tube formation of c‑Kit cells following
stimulation of conditioned medium from H9c2 cells treated with VEGF‑B.
GHL has made substantial contributions to conception and design of the
study, carried out the cell experimental studies, and participated in animal
experimental and drafted the manuscript. YXL carried out animal experi‑
mental and cell autophagy assay.LB carried out animal experimental and cell
apoptosis assay. WMX carried out cell apoptosis assay. XYL participated in
heart function and infarction size assay. FZ carried out Western Blotting assay.
LW participated in cell culture. LZ carried out cell autophagy assay. JMT partici‑
pated in drafting and interpretation and has made substantial contributions
to conception and design of the study of data. JNW participated in drafting
and interpretation.SYC has made substantial contributions to conception
and critically revised the manuscript. XHH made substantial contributions to
conception and design of the study of data. All authors read and approved
the final manuscript.
This study was supported by grants from National natural Science Founda‑
tion of China (81170095 to J.M.T), Foundation of Hubei Department Science
(2014CFB644 to J.M.T), Hubei Health Department Science Foundation (JX5B24
to J.M.T), Hubei Education Department Science Foundation (T201112 to
J.M.T), and National Institutes of Health (HL093429 and HL107526 to S.Y.C.).
All authors declare that they have no competing interests.
1. Hausenloy DJ , Yellon DM . Myocardial ischemia‑reperfusion injury: a neglected therapeutic target . J Clin Invest . 2013 ; 123 : 92 - 100 .
2. Ferrara N , Gerber HP , Le Couter J. The biology of VEGF and its receptors . Nat Med . 2003 ; 9 : 669 - 76 .
3. Tang JM , Wang JN , Zhang L , Zheng F , Yang JY , Kong X , Guo LY , Chen L , Huang YZ , Wan Y , Chen SY . VEGF/SDF‑1 promotes cardiac stem cell mobilization and myocardial repair in the infarcted heart . Cardiovasc Res . 2011 ; 91 : 402 - 11 .
4. Kajdaniuk D , Marek B , Borgiel‑Marek H , Kos‑Kudła B. Vascular endothe ‑ lial growth factor (VEGF)-part 1: in physiology and pathophysiology . Endokrynol Pol . 2011 ; 62 : 444 - 55 .
5. Lähteenvuo JE , Lähteenvuo MT , Kivelä A , Rosenlew C , Falkevall A , Klar J , Heikura T , Rissanen TT , Vähäkangas E , Korpisalo P , Enholm B , Carmeliet P , Alitalo K , Eriksson U , Ylä‑Herttuala S . Vascular endothelial growth factor ‑B induces myocardium‑specific angiogenesis and arteriogenesis via vascular endothelial growth factor receptor‑1‑ and neuropilin receptor ‑ 1‑ dependent mechanisms . Circulation . 2009 ; 119 : 845 - 56 .
6. Olofsson B , Pajusola K , Kaipainen A , von Euler G , Joukov V , Saksela O , Orpana A , Pettersson RF , Alitalo K , Eriksson U. Vascular endothelial growth factor B, a novel growth factor for endothelial cells . Proc Natl Acad Sci USA . 1996 ; 93 : 2576 - 81 .
7. Aase K , Lymboussaki A , Kaipainen A , Olofsson B , Alitalo K , Eriksson U. Localization of VEGF‑B in the mouse embryo suggests a paracrine role of the growth factor in the developing vasculature . Dev Dyn . 1999 ; 215 : 12 - 25 .
8. Karpanen T , Bry M , Ollila HM , Seppänen‑Laakso T , Liimatta E , Leskinen H , Kivelä R , Helkamaa T , Merentie M , Jeltsch M , Paavonen K , Andersson LC , Mervaala E , Hassinen IE , Ylä‑Herttuala S , Oresic M , Alitalo K. Overex ‑ pression of vascular endothelial growth factor‑B in mouse heart alters cardiac lipid metabolism and induces myocardial hypertrophy . Circ Res . 2008 ; 103 : 1018 - 26 .
9. Hagberg CE , Falkevall A , Wang X , Larsson E , Huusko J , Nilsson I , van Meeteren LA , Samen E , Lu L , Vanwildemeersch M , Klar J , Genove G , Pietras K , Stone‑Elander S , Claesson‑ Welsh L , Ylä‑Herttuala S , Lindahl P , Eriksson U . Vascular endothelial growth factor B controls endothelial fatty acid uptake . Nature . 2010 ; 464 : 917 - 21 .
10. Hagberg CE , Mehlem A , Falkevall A , Muhl L , Fam BC , Ortsäter H , Scotney P , Nyqvist D , Samén E , Lu L , Stone‑Elander S , Proietto J , Andrikopoulos S , Sjöholm A , Nash A , Eriksson U . Targeting VEGF‑B as a novel treatment for insulin resistance and type 2 diabetes . Nature . 2012 ; 490 : 426 - 30 .
11. Kivelä R , Bry M , Robciuc MR , Räsänen M , Taavitsainen M , Silvola JM , Saraste A , Hulmi JJ , Anisimov A , Mäyränpää MI , Lindeman JH , Eklund L , Hellberg S , Hlushchuk R , Zhuang ZW , Simons M , Djonov V , Knuuti J , Mervaala E , Alitalo K. VEGF‑B‑induced vascular growth leads to metabolic reprogramming and ischemia resistance in the heart . EMBO Mol Med . 2014 ; 6 : 307 - 21 .
12. Huusko J , Lottonen L , Merentie M , Gurzeler E , Anisimov A , Miyanohara A , Alitalo K , Tavi P , Ylä‑Herttuala S. AAV9‑mediated VEGF‑B gene transfer improves systolic function in progressive left ventricular hypertrophy . Mol Ther . 2012 ; 20 : 2212 - 21 .
13. Zhao T , Zhao W , Chen Y , Liu L , Ahokas RA , Sun Y. Differential expression of vascular endothelial growth factor isoforms and receptor subtypes in the infarcted heart . Int J Cardiol . 2013 ; 167 : 2638 - 45 .
14. Devaux Y , Vausort M , Azuaje F , Vaillant M , Lair ML , Gayat E , Lassus J , Ng LL , Kelly D , Wagner DR , Squire IB . Low levels of vascular endothelial growth factor B predict left ventricular remodeling after acute myocardial infarction . J Card Fail . 2012 ; 18 : 330 - 7 .
15. Zentilin L , Puligadda U , Lionetti V , Zacchigna S , Collesi C , Pattarini L , Ruozi G , Camporesi S , Sinagra G , Pepe M , Recchia FA , Giacca M. Cardiomyocyte VEGFR‑1 activation by VEGF‑B induces compensatory hypertrophy and preserves cardiac function after myocardial infarction . FASEB J . 2010 ; 24 : 1467 - 78 .
16. Tillmanns J , Rota M , Hosoda T , Misao Y , Esposito G , Gonzalez A , Vitale S , Parolin C , Yasuzawa‑Amano S , Muraski J , De Angelis A , Lecapitaine N , Siggins RW , Loredo M , Bearzi C , Bolli R , Urbanek K , Leri A , Kajstura J , Anversa P . Formation of large coronary arteries by cardiac progenitor cells . Proc Natl Acad Sci USA . 2008 ; 105 : 1668 - 73 .
17. D'Amario D , Leone AM , Iaconelli A , Luciani N , Gaudino M , Kannappan R , Manchi M , Severino A , Shin SH , Graziani F , Biasillo G , Macchione A , Smaldone C , De Maria GL , Cellini C , Siracusano A , Ottaviani L , Massetti M , Goichberg P , Leri A , Anversa P , Crea F . Growth properties of cardiac stem cells are a novel biomarker of patients' outcome after coronary bypass surgery . Circulation . 2014 ; 129 : 157 - 72 .
18. Dhondt J , Peeraer E , Verheyen A , Nuydens R , Buysschaert I , Poesen K , Van Geyte K , Beerens M , Shibuya M , Haigh JJ , Meert T , Carmeliet P , Lambrechts D . Neuronal FLT1 receptor and its selective ligand VEGF‑B protect against retrograde degeneration of sensory neurons . FASEB J . 2011 ; 25 : 1461 - 73 .
19. Huang GQ , Wang JN , Tang JM , Zhang L , Zheng F , Yang JY , Guo LY , Kong X , Huang YZ , Liu Y , Chen SY . The combined transduction of copper, zinc‑superoxide dismutase and catalase mediated by cell‑penetrating peptide, PEP‑1, to protect myocardium from ischemia‑reperfusion injury . J Transl Med . 2011 ; 9 : 73 .
20. Tang JM , Luo B , Xiao JH , Lv YX , Li XL , Zhao JH , Zheng F , Zhang L , Chen L , Yang JY , Guo LY , Wang L , Yan YW , Pan YM , Wang JN , Li DS , Wan Y. Chen SY . VEGF‑A promotes cardiac stem cell engraftment and myocardial repair in the infarcted heart . Int J Cardiol . 2015 ; 183 : 221 - 31 .
21. Falk T , Yue X , Zhang S , McCourt AD , Yee BJ , Gonzalez RT , Sherman SJ . Vascular endothelial growth factor‑B is neuroprotective in an in vivo rat model of Parkinson's disease . Neurosci Lett . 2011 ; 496 : 43 - 7 .
22. Zhang L , Dong XW , Wang JN , Tang JM , Yang JY , Guo LY , Zheng F , Kong X , Huang YZ , Chen SY . PEP‑1 ‑ CAT‑transduced mesenchymal stem cells acquire an enhanced viability and promote ischemia‑induced angiogenesis . PLoS One . 2012 ; 7 : e52537 .
23. Hou Q , Hsu YT . Bax translocates from cytosol to mitochondria in cardiac cells during apoptosis: development of a GFP‑Bax ‑stable H9c2 cell line for apoptosis analysis . Am J Physiol Heart Circ Physiol . 2005 ; 289 : H477 - 87 .
24. Bry M , Kivelä R , Leppänen VM , Alitalo K. Vascular endothelial growth factor‑B in physiology and disease . Physiol Rev . 2014 ; 94 : 779 - 94 .
25. Wang X , Liu J , Zhen J , Zhang C , Wan Q , Liu G , Wei X , Zhang Y , Wang Z , Han H , Xu H , Bao C , Song Z , Zhang X , Li N , Yi F . Histone deacetylase 4 selectively contributes to podocyte injury in diabetic nephropathy . Kidney Int . 2014 ; 86 : 712 - 25 .
26. la de Cruz‑Morcillo MA , Valero ML , Callejas‑ Valera JL , Arias‑ González L , Melgar‑Rojas P , Galán‑Moya EM , García‑ Gil E , García‑ Cano J , SánchezPrieto R . P38MAPK is a major determinant of the balance between apoptosis and autophagy triggered by 5‑fluorouracil: implication in resistance . Oncogene . 2012 ; 31 : 1073 - 85 .
27. Li P , Shi J , He Q , Hu Q , Wang YY , Zhang LJ , Chan WT , Chen WX . Streptococcus pneumoniae induces autophagy through the inhibition of the PI3K‑I/ Akt/mTOR pathway and ROS hypergeneration in A549 cells . PLoS One . 2015 ; 10 : e0122753 .
28. Serpi R , Tolonen AM , Huusko J , Rysä J , Tenhunen O , Ylä‑Herttuala S , Ruskoaho H . Vascular endothelial growth factor‑B gene transfer prevents angiotensin II‑induced diastolic dysfunction via proliferation and capillary dilatation in rats . Cardiovasc Res . 2011 ; 89 : 204 - 13 .
29. McLean BA , Kienesberger PC , Wang W , Masson G , Zhabyeyev P , Dyck JR , Oudit GY . Enhanced recovery from ischemia‑reperfusion injury in PI3Kα dominant negative hearts: investigating the role of alternate PI3K isoforms, increased glucose oxidation and MAPK signaling . J Mol Cell Cardiol . 2013 ; 54 : 9 - 18 .
30. Lakkisto P , Kytö V , Forsten H , Siren JM , Segersvärd H , Voipio‑Pulkki LM , Laine M , Pulkki K , Tikkanen I. Heme oxygenase‑1 and carbon monoxide promote neovascularization after myocardial infarction by modulating the expression of HIF‑1alpha, SDF‑1alpha and VEGF‑B. Eur J Pharmacol . 2010 ; 635 : 156 - 64 .
31. Ren B , Deng Y , Mukhopadhyay A , Lanahan AA , Zhuang ZW , Moodie KL , Mulligan‑Kehoe MJ , Byzova TV , Peterson RT . Simons M. ERK1/2‑Akt1 crosstalk regulates arteriogenesis in mice and zebrafish . J Clin Invest . 2010 ; 120 : 1217 - 28 .
32. Hassan MH , Othman EE , Hornung D , Al‑Hendy A . Gene therapy of benign gynecological diseases . Adv Drug Deliv Rev . 2009 ; 61 : 822 - 35 .
33. Rose BA , Force T , Wang Y . Mitogen‑activated protein kinase signaling in the heart: angels versus demons in a heart‑breaking tale . Physiol Rev . 2010 ; 90 : 1507 - 46 .
34. Sussman MA , Völkers M , Fischer K , Bailey B , Cottage CT , Din S , Gude N , Avitabile D , Alvarez R , Sundararaman B , Quijada P , Mason M , Konstandin MH , Malhowski A , Cheng Z , Khan M , McGregor M. Myocardial AKT : the omnipresent nexus . Physiol Rev . 2011 ; 91 : 1023 - 70 .
35. Fujio Y , Walsh K. Akt mediates cytoprotection of endothelial cells by vascular endothelial growth factor in an anchorage‑ dependent manner . J Biol Chem . 1999 ; 274 : 16349 - 54 .
36. Gerber HP , McMurtrey A , Kowalski J , Yan M , Keyt BA , Dixit V , Ferrara N. Vascular endothelial growth factor regulates endothelial cell survival through the phosphatidylinositol 3′‑kinase/Akt signal transduction pathway. Requirement for Flk‑1/KDR activation . J Biol Chem . 1998 ; 273 : 30336 - 43 .
37. Ma S , Wang Y , Chen Y , Cao F . The role of the autophagy in myocardial ischemia/reperfusion injury . Biochim Biophys Acta . 2015 ; 1852 : 271 - 6 .
38. Evans‑Anderson HJ , Alfieri CM , Yutzey KE . Regulation of cardiomyocyte proliferation and myocardial growth during development by FOXO transcription factors . Circ Res . 2008 ; 102 : 686 - 94 .
39. van Berlo JH , Kanisicak O , Maillet M , Vagnozzi RJ , Karch J , Lin SC , Middleton RC , Marbán E , Molkentin JD . C‑kit+ cells minimally contribute cardiomyocytes to the heart . Nature . 2014 ; 509 : 337 - 41 .