Autophagy mediates the beneficial effect of hypoxic preconditioning on bone marrow mesenchymal stem cells for the therapy of myocardial infarction
Zhang et al. Stem Cell Research & Therapy
Autophagy mediates the beneficial effect of hypoxic preconditioning on bone marrow mesenchymal stem cells for the therapy of myocardial infarction
Zheng Zhang 0
Zhitao Jin 0
Liping Ding 0
Wei Jiang 0
Junke Yang 0
Feng Cao 1
Taohong Hu 0
0 Department of Cardiology, The General Hospital of the PLA Rocket Force , Beijing 100088 , China
1 Department of Cardiology, The General Hospital of Chinese People's Liberation Army , Beijing 100853 , China
Background: Stem cell therapy has emerged as a promising therapeutic strategy for myocardial infarction (MI). However, the poor viability of transplanted stem cells hampers their therapeutic efficacy. Hypoxic preconditioning (HPC) can effectively promote the survival of stem cells. The aim of this study was to investigate whether HPC improved the functional survival of bone marrow mesenchymal stem cells (BM-MSCs) and increased their cardiac protective effect. Methods: BM-MSCs, isolated from Tg(Fluc-egfp) mice which constitutively express both firefly luciferase (Fluc) and enhanced green fluorescent protein (eGFP), were preconditioned with HPC (1% O2) for 12 h, 24 h, 36 h, and 48 h, respectively, followed by 24 h of hypoxia and serum deprivation (H/SD) injury. Results: HPC dose-dependently increased the autophagy in BM-MSCs. However, the protective effects of HPC for 24 h are most pronounced. Moreover, hypoxic preconditioned BM-MSCs (HPCMSCs) and nonhypoxic preconditioned BM-MSCs (NPCMSCs) were transplanted into infarcted hearts. Longitudinal in vivo bioluminescence imaging (BLI) and immunofluorescent staining revealed that HPC enhanced the survival of engrafted BM-MSCs. Furthermore, HPCMSCs significantly reduced fibrosis, decreased apoptotic cardiomyocytes, and preserved heart function. However, the beneficial effect of HPC was abolished by autophagy inhibition with 3-methyladenine (3-MA) and Atg7siRNA. Conclusion: This study demonstrates that HPC may improve the functional survival and the therapeutic efficiencies of engrafted BM-MSCs, at least in part through autophagy regulation. Hypoxic preconditioning may serve as a promising strategy for optimizing cell-based cardiac regenerative therapy.
Hypoxic preconditioning; Mesenchymal stem cells; Myocardial infarction; Apoptosis; Autophagy
Myocardial infarction (MI) and consequent heart failure
remain the leading cause of morbidity and mortality
worldwide. The traditional therapies, aimed at preserving
the function of the remaining cardiac myocytes, are
palliative . Currently, stem cell therapy has emerged as a
promising therapeutic strategy to effectively reverse
cardiac damage and restore cardiac function in terms of
its potential contribution to cardiovascular regeneration
[2, 3]. Among these cells, bone marrow mesenchymal
stem cells (BM-MSCs), capable of self-renewal and
differentiation into various mesenchymal tissues, have
been considered as an optimal candidate in regenerative
medicine. A large number of studies have demonstrated
that BM-MSC transplantation can effectively attenuate
myocardial injury and improve cardiac function [4, 5].
In practical applications, however, the therapeutic
efficiency of stem cell transplantation is greatly limited by
© The Author(s). 2017 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
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the poor viability and function of donor cells . The
harsh host ischemic microenvironment in the ischemic
myocardium resulted in high levels of apoptosis, which
further impaired the functional survival of transplanted
donor stem cells. Moreover, massive cell death not only
hampers the efficiency of the therapy, but also introduces
an additional burden to the infarcted myocardium [7, 8].
Therefore, optimized strategies that enhance the cell
viability and retention of transplanted donor cells are
crucial to improving the efficiency of MSC-mediated
therapy for MI.
Previous in vitro and in vivo studies have demonstrated
that sublethal hypoxic preconditioning (HPC)
significantly increases the expressions of pro-survival and
pro-angiogenic cellular factors in MSCs [9, 10].
Moreover, HPC increases cell survival, inhibits extensive
apoptosis, and thereby improves the adaptability of
MSCs to hypoxic injury . Therefore, HPC has been
considered as a promising adjuvant strategy in the
cellbased treatment for MI. However, the optimal HPC
protocol of BM-MSCs for MI therapy remains
uncertain. Furthermore, detailed mechanisms underlying the
beneficial effects of HPC have not been elucidated.
Autophagy is a highly conserved catabolic process to
degrade damaged organelles for recycling of cytoplasmic
components by forming autophagosomes, which then
fuse with lysosomes to form autolysosomes . The
evidence has demonstrated that this can either protect
cells or contribute to cell death depending on the
different cell types and intensity of stimulus . Autophagy
at basal levels is involved in development, differentiation,
and maintaining normal function in various organisms.
Hence, autophagy has been generally considered as a
protective cellular response against various stresses, such
as starvation or nutrient depletion. Conversely, previous
studies also suggested that extensive and prolonged
autophagy may be a promoter of apoptosis, leading to
cell death as type II programmed cell death . A
significant body of studies has demonstrated that HPC can
induce autophagy in MSCs. Moreover, Wang et al. 
revealed that modest HPC can offer neuroprotection by
activating autophagic pathways, indicating a
neuroprotective role of autophagy in HPC. Despite expansion of
our knowledge, the detailed effect of HPC on autophagy
of MSCs has not been fully understood. Accordingly, we
hypothesized that the protective effects of HPC on
BMMSCs are mediated by regulating autophagy.
L2G85 transgenic mice were created on the FVB
background which stably express both firefly luciferase
(Fluc) and enhanced green fluorescence protein (eGFP;
fLuc-eGFP) in all tissues and organs. Mice were housed
in a temperature-controlled animal facility with a 12-h
light/dark cycle, with tap water and rodent chow
provided ad libitum. All animal studies were performed
via a protocol approved by the Animal Care and Use
Committee of the Second Artillery General Hospital of
PLA (approval ID: 5034) and were in compliance with
the Guidelines for the Care and Use of Laboratory
Animals, as published by the National Academy Press.
Isolation, culture, and identification of BM-MSCs from
L2G85 transgenic mice
BM-MSCs were isolated and expanded with a modified
procedure as described previously . In brief, bone
marrow was flushed from the femoral and tibia of adult
L2G85 mice with fetal bovine serum (FBS)-free Dulbecco’s
modified Eagle’s medium (DMEM). After passing through
a 70-μm strainer and centrifugation at 1200 rpm for 5
min at room temperature, the cell pellet was resuspended
in DMEM supplemented with 20% FBS and incubated at
37 °C in an atmosphere containing 5% CO2. After 24 h,
the culture medium was replaced to remove the
nonadherent cells, and then was completely replaced every 3
days. Third-passage BM-MSCs with optimal growth at the
third generation were used for different treatments to
avoid contamination with other cell types.
BM-MSCs were characterized as CD44+, CD90+,
CD34–, and CD45– using cytofluorimetric analysis as
described previously . Briefly, after being incubated with
1 μL monoclonal PE-conjugated antibodies against
specific membrane markers (CD44, CD90, CD34, and
CD45; BD, San Jose, CA, USA) for 1 h, BM-MSCs were
processed through a FACS Calibur system (BD, San Jose,
CA, USA) according to the manufacturer’s protocol.
Cells were gated according to their high fluorescence.
Furthermore, the multipotency of BM-MSCs was
confirmed by induction of osteogenic and adipogenic
differentiation as previously described [8, 16]. Adipogenic
medium (alpha-modified Eagles’s medium (αMEM) with
10% fetal calf serum (FCS), 1% antibiotics, 50 μM
indomethacin (Sigma), 0.5 mM IBMX (Sigma), and 1 μM
dexamethasone) and osteogenic differentiation of
BMMSCs was induced by culturing cells in osteogenic
medium (OM; 10% FBS, 0.1 μM dexamethasone, 10 mM
β-glycerophosphate, and 0.2 mM ascorbic acid in
αMEM) were added to the confluent layer of BM-MSCs for
21 days, respectively. The adipogenic cells were stained
with a working solution of oil red O for 10–15 min at
room temperature. Meanwhile, the degree of
extracellular matrix calcification was estimated using an alizarin
red S stain.
Hypoxic preconditioning and ischemic injury
Ischemic injury for the BM-MSCs was stimulated with
hypoxia/serum deprivation (H/SD) injury as described
previously . Briefly, after being replaced in
glucosefree DMEM without FBS, BM-MSCs were exposed to
hypoxia (94% N2/5% CO2/1% O2) in an anaerobic system
(Thermo Forma) at 37 °C for 24 h. Meanwhile, BM-MSCs
incubated under normoxic conditions (37 °C in 95% air,
5% CO2) with full medium for equivalent periods were
used as a control. Moreover, BM-MSCs were subjected to
an atmosphere of 1% O2 and 5% CO2, while kept in full
medium for HPC.
Autophagy inhibition and autophagy evaluation
To study the effect of autophagy on the hypoxia injury
of BM-MSCs, 3-methyladenine (3MA; 5 mM, Sigma)
was administrated for 1 h to inhibit autophagy.
Consistent with 3-MA, Atg 7 gene silencing by small interfering
RNAs (siRNAs) further suppressed the formation of
autophagic vacuoles as described previously . Briefly,
BM-MSCs were plated on to six-well plates (1 × 105 cells
per cm2) for 24 h before transfection. Cells were
transiently transfected with siRNAs targeting Atg7 or control
siRNAs (Cell signal) using Lipofectamine™ 2000 according
to the manufacturer’s protocol. All of these treatments
were performed in duplicate.
The autophagy of BM-MSCs was measured by green
fluorescence protein (GFP)-LC3 fusion protein, a widely
accepted marker to visualize formation of autophagic
vacuoles. Briefly, Lipofectamine LTX and plasmid DNA were
administrated to the culture system for 4 h at 37 °C.
GFPLC3 puncta in BM-MSCs were quantified by fluorescence
microscopy after different treatments. Five random fields
were counted and the percentages of cells with GFP-LC3
punctate were calculated. Meanwhile, the expressions of
LC-3, Beclin-1, and P62 were assessed by Western blot
assay. Furthermore, autophagosomes in BM-MSCs were
detected by transmission electron microscopy. Briefly,
after washing with phosphate-buffered saline (PBS) and
dehydration through graded ethanol, cells were
embedded in epoxy resin. Ultrathin sections were prepared
and stained with uranyl acetate (1%) and lead citrate
(0.2%). Images were recorded using a transmission
electron microscope (JEM1230; JEOL). The average
numbers of the autophagic structures in the cytoplasm were
Cell viability assay
The cell viability of BM-MSCs was assessed by
3-(4,5dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide
(MTT) assay as described previously . Briefly,
BMMSCs were plated in 96-well plates at 1 × 105 cells/well.
After different treatments, BM-MSCs were incubated with
MTT solution (5 g/L, Sigma) at 37 °C for 4 h. The
medium was then removed and 200 μL dimethyl
sulphoxide (DMSO) was added to each well. The
absorbance was determined at a wavelength of 490 nm. Optical
density (OD) values for each group were detected in six
duplicate wells and their averages were calculated.
Furthermore, we also assessed cell viability with
bioluminescence imaging (BLI) using the IVIS Kinetic system
(Caliper, Hopkinton, MA, USA) . Briefly, MSCs were
plated in 24-well plates (5 × 104 per well). After different
treatments, cell culture media were removed. Cells were
incubated with D-Luciferin reporter probe (4.5 μg/mL)
and then measured using the IVIS Xenogen Kinetic
system (Caliper Life Sciences, USA), using the following
imaging parameters: binning, 4; F/stop 1; time, 1 min.
Bioluminescent signals were analyzed using Living Image
4.0 software (Caliper, MA, USA) and quantified as
photons per second per centimeter square per steridian
Measurement of VEGF, bFGF, IGF-1, and HGF
The concentrations of vascular endothelial growth factor
(VEGF), basic fibroblast growth factor (bFGF), insulin-like
growth factor (IGF)-1, and hepatocyte growth factor
(HGF) secreted by MSCs were determined by
enzymelinked immunosorbent assay (ELISA) according to the
manufacturer’s instructions. All samples and standards
were measured in duplicate. In addition, paracrine
secretion with and without autophagy inhibition and different
HPC protocols were also evaluated.
MSC apoptosis was determined by terminal
deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)
assay using an assay kit (In Situ Cell Death Detection Kit;
Roche Diagnostics) according to the manufacturer’s
instructions . Briefly, after different treatments, BM-MSCs
were incubated with TdT and fluorescein-labeled dUTP for
45 min at 37 °C followed by 4,6-diamidino-2-phenylindole
(DAPI) for the identification of nuclei. Photographs were
taken using a confocal microscope (Olympus Fluoview
2000). For each group, five random fields were counted to
calculate the percentage of apoptotic cells. Meanwhile,
caspase-3 activity was measured using a Caspase-3 Assay
kit (Clontech, Mountain View, CA, USA) according to the
manufacturer’s instructions. All of these assays were
performed in a blinded manner.
Western blot assay
Western blotting was performed following a standard
protocol. Equal amounts of protein (50 μg/lane) were
separated by electrophoresis on 12% SDS-PAGE gels in a
Tris/HCl buffer system for 90 min at 120 V, and
sequentially electrophoretically transferred to nitrocellulose (NC)
membranes. After blocking with 5% nonfat dry milk (BD
Biosciences) at room temperature for 1 h, membranes
were subjected to immunoblotting with primary
antibodies overnight at 4 °C. After incubation with the
appropriate secondary antibody conjugated with horseradish
peroxidase, blot bands were visualized with an enhanced
chemiluminescence system (Amersham Bioscience).
Densitometric analysis of Western blots was performed
using VisionWorks LS, version 6.7.1. The following
primary antibodies were used: rabbit anti-mice LC-3 (1:500,
Cell Signaling Technology), rabbit anti-mice P62 (1:500,
Cell Signaling Technology), rabbit anti-mice Beclin-1
(1:500, Cell Signaling Technology), and rabbit anti-mice
β-actin (1:2000, Abcam).
Myocardial infarction model and MSC transplantation
MI was induced in adult C57BL/6 mice by permanent
ligation of the left anterior descending (LAD) artery as
described previously [7, 18]. In brief, mice were
anesthetized with isoflurane and mechanically ventilated.
Left thoracotomy was performed and the pericardium
was opened. The LAD artery was permanently ligated
with a 6-0 suture. The ligation was deemed successful
when the anterior wall of the left ventricle (LV) turned
pale and characteristic electrocardiographic (ECG)
changes were seen. Sham-operated control mice
underwent the same surgical procedures except that
the suture placed under the left coronary artery was
BM-MSC transplantation was performed immediately
after MI. After HPC for 24 h and 48 h, respectively, the
cells were collected and randomly divided into the
different groups separately. The suspended BM-MSCs (1 × 106)
were injected directly into the peri-infarcted areas
(multiple injections within the presumed infarct and
the border zone) using a Hamilton syringe with a
29gauge needle (in 20 mice in every group).
In vivo BLI of transplanted MSCs
BLI was performed to track engrafted MSCs using an
IVIS Kinetic system (Caliper, Hopkinton, MA, USA) as
described previously . After intraperitoneal injection
with D-luciferin (375 mg/kg body weight), recipient mice
were anesthetized with isoflurane and imaged for 10 min
on days 1, 3, 5, and 7, and weekly until sacrificed. Peak
signals (photons/s/cm2/sr) from a fixed region of interest
(ROI) were analyzed using Living Image® 4.0 software
(Caliper, MA, USA).
Postmortem histological assays
To evaluate the survival of MSCs in ischemic
myocardium, five mice were sacrificed 2 weeks after MSC
transplantation. The hearts were harvested and rapidly (within
a minute) fixed in 4% paraformaldehyde. Serial sections
were prepared at 5 μm thickness and stained with an
FITC-labeled anti-eGFP antibody and DAPI. Meanwhile,
cardiomyocytes were stained with an anti-cTn I antibody.
Cell engraftment was confirmed by identification of GFP
expression under fluorescent microscopy. The numbers of
GFP-positive cells and DAPI in each slide were calculated.
The data were expressed as the percentage of GFP+/DAPI
in five slides obtained from five frozen sections. To
validate the proliferation of engrafted MSCs,
immunofluorescence staining for the cell cycle-associated nuclear protein
Ki67 was performed. The numbers of GFP and ki67
double-positive cells were calculated. Furthermore, the
apoptosis of engrafted MSCs and myocardial cells were
determined by TUNEL assay as previously described.
TUNEL staining was performed with fluorescein-dUTP
(In Situ Cell Death Detection Kit; Roche Diagnostics) for
apoptotic cell nuclei and DAPI (Sigma) to stain all cell
nuclei. Additional staining for troponin I or GFP was
performed for the identification of myocardium and
transplanted BM-MSCs, respectively. The numbers of GFP and
TUNEL double-positive cells were calculated. In addition,
the percentage of apoptotic cardiomyocytes was termed
the apoptotic index. All of these assays were performed in
a blinded manner.
Evaluation of fibrosis
Masson’s trichrome staining was performed to assess the
infarct size. Recipient mice were sacrificed for
histological assay at 4 weeks after MI and MSC
transplantation . Hearts were fixed in 4% paraformaldehyde
and embedded in paraffin. Serial sections were prepared
at 5 μm thickness. Masson's trichrome stain was used to
detect fibrosis in the cardiac muscle. Ten anterolateral
sections from each heart were evaluated in their entirety
and quantified. The fibrosis area was determined by
measuring the collagen area as a proportion of the total
area using Imaging Pro Plus software.
Cardiac function was measured 7 days after MI by
transthoracic echocardiography at 2 days, 7 days, and weekly
until sacrifice at 4 weeks post-operation using a 30-MHz
transducer on a Vevo® 2100 ultrasound system
(VisualSonics, CA, USA) [18, 19]. Briefly, the mice were anesthetized
(2% isoflurane and oxygen) and put in a supine position.
Both two-dimensional and M-mode images were recorded
by a blinded investigator. The left ventricular end-systolic
volume (LVESV) and diameter (LVESD) and left ventricular
end-diastolic volume (LVEDV) and diameter (LVEDD)
were measured to calculate left ventricular ejection fraction
(LVEF) and fractional shortening (FS) via the following
equations: LVEF = (LVEDV – LVESV)/LVEDV × 100%,
LVFS = (LVEDD –LVESD)/LVEDD × 100%.
The results are presented as means ± SEM. Statistics
were calculated using Prism 5.0 (GraphPad Software
Inc, San Diego, CA, USA). Linear regression analysis
was performed to determine the correlation between
two variables. Statistical comparisons for different
groups were performed using either the Student’s t test
or one-way analysis of variance (ANOVA). p values
<0.05 were considered statistically significant.
Characterization of BM-MSCFluc+GFP+
BM-MSCs were characterized by in vitro multilineage
differentiation and flow cytometry analysis. Flow
cytometry results revealed that BM-MSCFluc+GFP+ were
uniformly positive for the MSC markers CD44, CD90,
and CD29, and negative for CD31, CD45, and CD34
(Fig. 1a). As shown in the representative images in
Fig. 1b, 70% of BM-MSCFluc+GFP+ had an adipocyte
phenotype after adipogenic medium incubation for 21
days, which was assessed by oil red O staining.
Meanwhile, alizarin red S staining for calcium deposition
demonstrated that BM-MSCFluc+GFP+ could also differentiate
into osteogenic cells. These multilineage differentiation
results revealed the pluripotency of BM-MSCFluc+GFP+.
BM-MSCFluc+GFP+, isolated from Tg(Fluc-egfp) reporter
transgenic mice, constitutively express both Fluc and
eGFP. As shown in the representative images in Fig. 1b,
BM-MSCFluc+GFP+ exhibited fibroblast-like morphology
and expressed GFP. Furthermore, in vitro BLI
demonstrated a robust linear correlation between the number of
BM-MSCFluc+GFP+ and average Fluc radiance (r2 = 0.98;
Fig .1c), indicating that BLI of Fluc was could be reliably
used to monitor the viability of engrafted MSCFluc+GFP+
quantitatively in vivo.
Hypoxic preconditioning for 24 h increases the viability
and growth factor secretions of BM-MSCFluc+GFP+
H/SD treatment was performed to imitate ischemic
conditions in vitro. In order to develop the optimal HPC
protocol, MSCFluc+GFP+ were preconditioned by hypoxic
incubation (1% O2) in full medium for 12, 24, 36, or 48 h
prior to H/SD injury. BLI results displayed a remarkable
decline of BLI signal intensity in MSCFluc+GFP+ after H/SD
injury compared with normal control (3.07 ± 0.67 × 105 p/
s/cm2/sr after H/SD versus 10.48 ± 0.82 × 105 p/s/cm2/sr
Fig. 1 Characterization of BM-MSC Fluc+GFP+. a Flow cytometry results show that BM-MSCFluc+GFP+ were uniformly negative for CD31, CD34 and
CD45, and positive for CD44, CD29 and CD90. b The differentiation potential of BM-MSC Fluc+GFP+. Fibroblast-like shaped BM-MSCFluc+GFP+ were green
fluorescent protein (GFP) positive. Adipogenesis of BM-MSC Fluc+GFP+ under the adipogenic differentiation conditions was detected by oil red O
staining. Osteogenesis was evaluated by alizarin red S staining (scale bar = 100 μm). c Ex vivo BLI shows a linear relationship between cell
number and Fluc reporter gene activity
under normal conditions; p < 0.05) (Fig. 2a and b).
Moreover, the impaired viability of MSCFluc+GFP+ after
H/SD injury was ameliorated by HPC for 24 h (8.18 ±
0.53 × 105 p/s/cm2/sr versus 3.07 ± 0.67 × 105 p/s/cm2/sr
for H/SD; p < 0.05). Meanwhile, HPC for 12 h seemed
to enhance the viability of BM-MSCs compared with the
H/SD group, but without statistical significance (3.87 ±
0.47 × 105 p/s/cm2/sr versus 3.07 ± 0.67 × 105 p/s/cm2/sr
Fig. 2 Effect of hypoxic preconditioning (HPC) on the viability and paracrine mechanism of BM-MSC Fluc+GFP+. a Representative in vitro BLI results of
BM-MSCFluc+GFP+ with HPC for 0 h, 12 h, 24 h, 36 h, and 48 h under normal conditions and after hypoxia/serum deprivation (H/SD). b The quantification
of BLI assays. c MTT assay demonstrated the effects of HPC (0 h, 12 h, 24 h, 36 h, and 48 h) on viability of BM-MSCFluc+GFP+. d ELISA assay demonstrated
the levels of vascular endothelial growth factor (VEGF) (d), basic fibroblast growth factor (bFGF) (e), insulin-like growth factor-1 (IGF-1) (f), and
hepatocyte growth factor (HGF) (g) within BM-MSCFluc+GFP+ supernatants. Data are expressed as the mean ± SEM; n = 5; *p < 0.05
for H/SD; p > 0.05). However, HPC for 36 h and 48 h had
no protective effect on the viability of BM-MSCs
subjected to H/SD injury. In addition, MTT results also
confirmed that HPC for 24 h protected the impaired viability
of MSCFluc+GFP+ after H/SD injury (1.23 ± 0.04 versus
0.48 ± 0.02 for H/SD; p < 0.05; Fig. 2c). However, there
was no effect of HPC for 12, 36, or 48 h on the viability of
BM-MSCs after H/SD injury (p > 0.05; Fig. 2c).
It has been shown that MSCs contribute to cardiac
repair and regeneration at least in part by a paracrine
mechanism. Therefore, we evaluated the effect of HPC on
cytokine secretion in MSCFluc+GFP+ by ELISA assays. As
shown in Fig. 2d–g, HPC for 24 h significantly increased
VEGF, bFGF, IGF-1, and HGF secreted by MSCs.
However, these paracrine secretion levels were decreased
after administration of 3-MA and Atg7 siRNA (Additional
file 1: Figure S1). Furthermore, HPC for 48 h had a
downregulation effect on VEGF, bFGF, IGF-1, and HGF
secreted by MSCs. However, those paracrine secretion
levels were dramatically elevated after administration of
3-MA and Atg7 siRNA (Additional file 2: Figure S2).
The apoptosis of BM-MSCFluc+GFP+ was decreased by
hypoxic preconditioning for 24 h while increased by
hypoxic preconditioning for 48 h
To analyze the anti-apoptotic effect of HPC, TUNEL
assay and caspase-3 activity assay were performed to
evaluate the apoptosis of BM-MSCs induced by H/SD.
Representative immunofluorescence images and
quantitative analyses are shown in Fig. 3a and b. H/SD injury
increased the TUNEL-positive cells (27. 32 ± 1.41%
versus 5.63 ± 0.72% for normal; p < 0.05), which was
decreased by HPC for 24 h (14. 69 ± 1.13% versus 27. 32 ±
1.41% for H/SD; p < 0.05). Moreover, HPC for 12 h had
no effect on the apoptosis of BM-MSCs induced by H/SD
injury (p > 0.05). Interestingly, the percentage of
TUNELpositive cells in the HPC 36 h group was 36.15 ± 1.57%,
higher than that in the H/SD group without statistical
significance (p > 0.05). Furthermore, HPC for 48 h
significantly increased the apoptosis of BM-MSCs compared
with the H/SD group (44. 73 ± 1.63% versus 27. 32 ±
1.41% for H/SD; p < 0.05), which was reduced by 3-MA
and Atg7 siRNA (p < 0.05; Additional file 3: Figure S3).
Concurrently, we also found that the caspase-3 enzymatic
activity in the H/SD group was significantly increased
compared with that in the normal group (7.13 ± 0.19 × 103
RFU versus 3.58 ± 0.02 × 103 RFU for normal; p < 0.05)
(Fig. 3c). Furthermore, HPC for 24 h decreased the
enhanced caspase-3 enzymatic activity induced by H/SD
(5.72 ± 0.52 × 103 RFU versus 7.13 ± 0.19 × 103 RFU for H/
SD; p < 0.05). However, HPC for 48 h significantly
increased the caspase-3 enzymatic activities compared with
the H/SD group (8.22 ± 0.38 × 103 RFU versus 7.13 ±
0.19 × 103 RFU for H/SD; p < 0.05). Taken together, these
data suggested that HPC for 24 h prevented apoptosis of
BM-MSCs while HPC for 48 h promoted apoptosis of
BM-MSCs induced by H/SD injury.
Hypoxic preconditioning increases autophagy in BM-MSCs
To explore the autophagy level under hypoxic
conditions, BM-MSCs were transfected with GFP-LC3 to
demonstrate the LC3 expression. Meanwhile, the
protein expressions of LC3-I/LC3-II, P62, and Beclin-1
were also assessed by Western blot assay. Furthermore,
the autophagosomes in BM-MSCs were also detected
by transmission electron microscopy. Autophagosome
formation was increased by hypoxic preconditioning
time-dependently (Fig. 4a). Moreover, the
representative immunofluorescence microphotographs and the
quantitative analyses in Fig. 4c and d revealed that the
percentage of cells with punctate LC3 was 5.12 ± 0.07%
under normal conditions. Conversely, the percentages
of cells with punctate LC3 were increased by HPC
time-dependently. Furthermore, the representative
Western blot assay and semiquantitative analysis
revealed that HPC significantly increased the expressions
of LC3-II and Beclin-1 in BM-MSCs. However, the
levels of expression of p62 were decreased by HPC
(Fig. 4e–h). Taken together, these results suggested that
HPC activated autophagy in BM-MSCs.
Autophagy inhibition by 3-methyladenine and Atg7 siRNA
To determine the effect of autophagy on the protective
effect of HPC, autophagy in BM-MSCs was inhibited
by the autophagy inhibitor 3-MA and Atg7 siRNA,
respectively. The increased punctate LC3 formation
induced by HPC for 24 h was blocked by 3-MA and
Atg7 siRNA (Additional file 4: Figure S4).
Furthermore, quantitative analyses revealed that the
percentage of BM-MSCs with punctate LC3 in the 24-h HPC
group was 29.31 ± 4.72%, significantly higher than that
under normal condition (6.25 ± 1.14%; p < 0.05).
Conversely, the percentage of BM-MSCs with punctate
LC3 in the HPC + 3-MA group and HPC + Atg7
siRNA group was 12.32 ± 3.72% and 9.15 ± 0.79%,
respectively, significantly less than that in the HPC
group (p < 0.05). Collectively, these results indicated
that 3-MA and Atg7 siRNA were reliably able to
inhibit autophagy in BM-MSCs.
Autophagy contributes to the protective effect of HPC
against hypoxic stress
To gain an insight into the role of autophagy on the
protective effect of HPC against H/SD injury, we inhibited
Fig. 3 Effect of hypoxic preconditioning (HPC) on the apoptosis of BM-MSCs. a Representative immunofluorescence images of terminal
deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) (green fluorescence) and 4,6-diamidino-2-phenylindole (DAPI) (blue
fluorescence) in BM-MSCs under normal conditions and after hypoxia/serum deprivation (H/SD) (Scale bars = 20 μm). b Quantification of
the apoptotic BM-MSCs was presented as the percentage of apoptotic cells. c Histogram illustrated the caspase-3 enzymatic activity in
BM-MSCs in all groups. Data are expressed as means ± SEM; n = 5; *p < 0.05H/SD vs. Normal, #p<0.05
the autophagy in BM-MSCs by Atg7 siRNA and 3-MA.
Representative immunofluorescence images from the
TUNEL assay (Fig. 5a) revealed that HPC for 24 h
significantly decreased the apoptosis in BM-MSCs compared
with the H/SD group, which was abolished by 3-MA
treatment and Atg7 siRNA. Furthermore, quantitative analyses
(Fig. 5c) revealed that the percentage of
TUNELpositive cells in the hypoxia group was 33.73 ± 2.25%,
significantly higher than that in the normal group (5.76
± 1.12%; p < 0.05) and the HPC + H/SD group (18.35 ±
2.54%; p < 0.05). However, the percentages of
TUNELpositive BM-MSCs in the H/SD + HPC + 3-MA group and
the H/SD + HPC + Atg7 siRNA group were 35.14 ± 3.19%
and 32.31 ± 2.42%, respectively, significantly higher than
that in the HPC + H/SD group (18.35 ± 2.54%; p < 0.05).
Concurrently, BLI assays were performed to evaluate the
viability of BM-MSCs (Fig. 5b). Moreover, the quantitative
analyses (Fig. 5d) indicated that the impaired viability of
MSCs after H/SD injury was ameliorated by HPC for
24 h (8.23 ± 0.46 × 105 p/s/cm2/sr vs. 6.05 ± 0.38 × 05 p/
s/cm2/sr for H/SD; p < 0.05). However, autophagy
inhibition with 3-MA and Atg7 siRNA further abrogated
HPC-induced preservation of the viability of BM-MSCs
as manifested by the BLI signal (5.89 ± 0.43 × 105 p/s/
Fig. 4 Effect of hypoxic preconditioning (HPC) on the autophagy of BM-MSCs. a Representative electron micrographs revealed the autophagic
vacuole formation in BM-MSCs. b Quantification of the average numbers of the autophagic structures in the cytoplasm. c Autophagy flux was analyzed
in BM-MSCs infected with Ad-GFP-LC3 adenovirus. Representative immunofluorescence images of green fluorescent protein (GFP)-LC3 (green
fluorescence) and 4,6-diamidino-2-phenylindole (DAPI) (blue fluorescence) in BM-MSCs with HPC for the indicated time points. Scale bars = 20
μm. d Quantification of autophagy flux was presented as the percentage of BM-MSCs with punctate LC3 in all groups. e Representative
Western blots of LC3-I/LC3-II, Beclin-1, and P62 in BM-MSCs with HPC for 0 h, 12 h, 24 h, 36 h, and 48 h, respectively. Semiquantification of the
protein expressions of LC3-II (f), Beclin-1 (g), and p62 (h) at the indicated time points. Data are expressed as means ± SEM; n = 5; *p < 0.05
cm2/sr for HPC + H/SD + 3-MA, 5.12 ± 0.53 × 105 p/s/
cm2/sr for HPC + H/SD + Atg7 siRNA versus 8.23 ±
0.46 × 105 p/s/cm2/sr for H/SD + HPC; p < 0.05).
HPC for 24 h promotes survival of engrafted BM-MSCs
To determine the effect of HPC on the viability of
BMMSCs transplanted into infarcted hearts, BLI was
performed for 4 weeks. Representative BLI results and
the quantitative analyses revealed a progressive decay
of the BLI signal within 4 weeks after transplantation.
In contrast, HPC for 24 h facilitated the survival of
engrafted BM-MSCs, which was significantly obliterated
by autophagy inhibition with 3-MA and Atg7 siRNA
(Fig. 6a and b). Furthermore, HPC for 24 h facilitates the
survival of engrafted BM-MSCs, while HPC for 48 h
showed a sharply progressive decay of the BLI signal after
transplantation (Additional file 5: Figure S5).
To further confirm the in vivo BLI results for BM-MSC
survival, the hearts were harvested 2 weeks after cell
transplantation and stained for GFP. Cell survival was
evaluated by calculating the ratio GFP/DAPI. The
GFPpositive cells were more frequently observed in mice
Fig. 5 Autophagy modulated the protective effect of hypoxic preconditioning (HPC) on BM-MSCs. a Representative terminal deoxynucleotidyl
transferase-mediated nick-end labeling (TUNEL) images of BM-MSCs treated by HPC for 24 h with or without autophagy inhibition by 3-methyladenine
(3-MA) and Atg7 small interfering RNA (siRNA) (Scale bars = 20 μm). b Representative in vitro BLI results of BM-MSCsFluc+GFP+ in all groups. c The
quantification of the apoptotic BM-MSCs. d The quantification of the in vitro BLI assays. Data are expressed as means ± SEM; n = 5; *p < 0.05.
DAPI 4,6-diamidino-2-phenylindole, H/SD hypoxia/serum deprivation
administered with HPC MSCs (HPCMSCs). The ratio
GFP/DAPI in the HPCMSC group was 21.6 ± 4.3%,
significantly higher than that in the normal preconditioned
MSC (NPCMSC) group (12.2 ± 3.5%; p < 0.05) (Fig. 6d).
Furthermore, we also observed lower percentages of
TUNEL and GFP double-positive cells (7.3 ± 4.1%) in the
HPCMSC group compared with the NPCMSC group
(13.3 ± 3.9%; p < 0.05) (Fig. 6e). Additionally, the
proliferation of engrafted MSCs was detected by Ki-67
staining. The ratio of GFP and Ki67 double-positive cells in
the HPCMSC group was 30.5 ± 4.9%, significantly higher
than that in the NPCMSC group (12.3 ± 3.3%; p < 0.05)
(Fig. 6f ). These data suggest that HPC increases the
survival of BM-MSCs in post-infarct hearts. In contrast,
the beneficial effects of HPC on engrafted BM-MSCs
were significantly abolished by autophagy inhibition
with 3-MA and Atg7 siRNA.
Anti-apoptotic and pro-angiogenic effects facilitated by
HPC were abolished by autophagy inhibition
As shown by representative immunofluorescence
images in Fig. 7a, apoptotic cardiomyocytes, as
manifested by TUNEL positivity (in green), were more
frequently observed in the MI group than in the
sham-operated group (32.31 ± 2.48% versus 5.32 ±
1.03%; p < 0.05) (Fig. 7c). Moreover, transplantation of
Fig. 6 Evaluation of the survival of transplanted BM-MSCFluc+GFP+. a Representative longitudinal BLI spatiotemporally tracked BM-MSC Fluc
+GFP+ survival in normal preconditioned mesenchymal stem cells (NPCMSCs) (top row, n = 10), hypoxic preconditioned MSCs (HPCMSCs)
(second row, n = 10), the HPCMSCs + 3-methyladenine (3-MA) group (third row, n = 10), and the HPCMSCs + Atg7 small interfering RNA (siRNA)
group (bottom row, n = 10). Color scale bar values are in photons/s/cm2/sr. b Quantitative analysis of Firefly luciferase (Fluc) optical signals
on fixed regions of interest (ROI). c Representative confocal laser microscopic images of engrafted BM-MSCFluc+GFP+ (green fluorescent
protein (GFP): green fluorescence), cTnI (red, upper panel), terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL)
(red, middle panel), Ki67 (red, lower panel), and 4,6-diamidino-2-phenylindole (DAPI) (blue fluorescence) at 2 weeks after transplantation.
Scale bar = 50 μm. Quantitative analysis of the ratio of GFP/DAPI (d), GFP and TUNEL double-positive cells (GFP+TUNEL+/DAPI, e) and GFP
and Ki67 double-positive cells (GFP+Ki67+/DAPI, f) in all groups. Data are expressed as means ± SEM; n = 5; *p < 0.05
HPCBM-MSCs significantly decreased the apoptosis of
cardiomyocytes compared to the NPCBM-MSC group
(18.31 ± 2.75% versus 27.52 ± 1.72%; p < 0.05). However,
the percentages of TUNEL-positive cardiomyocytes in the
HPCMSC + 3-MA group and the HPCMSCs + Atg7siRNA
group were 31.87 ± 2.04% and 34.24 ± 2.21%,
respectively, significantly higher than that in the HPCMSC
group (18.31 ± 2.75%; p < 0.05).
Fig. 7 HPC facilitated the anti-apoptotic and pro-angiogenic effects of BM-MSCs. a Representative terminal deoxynucleotidyl transferase-mediated
nick-end labeling (TUNEL) images for cell apoptosis in the border zone around the infarcted area in mouse hearts. Apoptotic nuclei were identified as
TUNEL positive (green fluorescence). The myocardium was stained using a monoclonal antibody against cTnI (red fluorescence) and total nuclei by
4,6-diamidino-2-phenylindole (DAPI) counterstaining (blue fluorescence). Scale bar = 50 μm. b Capillaries in the infarct border zone were determined
by immunohistochemical staining for CD31-positive cells in all groups. Scale bars = 50 μm. Quantitative analysis of apoptotic nuclei (c) and capillaries in
the infarct border zone (d). Data are expressed as means ± SEM; n = 5; *p < 0.05. 3-MA 3-methyladenine, HPCMSCs hypoxic preconditioned mesenchymal
stem cells, HPF high-powered field, MI myocardial infarction, NPCMSCs normal preconditioned mesenchymal stem cells, Sham sham-operated, siRNA
small interfering RNA
Furthermore, we evaluated revascularization in the
peri-infarct zone (Fig. 7b and d). The vessel densities, as
evaluated by immunohistochemistry of CD31, were
reduced in the MI group compared to the
shamoperated group. Transplantation of HPCBM-MSCs
significantly increased the number of capillaries in CD31+
cells compared to the NPCBM-MSCs group. However,
the anti-apoptotic and the pro-angiogenic effects of
Hypoxia-preconditioned BM-MSCs reduce fibrosis and
preserved heart functional recovery after MI
To study the effects of HPC on the therapeutic efficiency of
engrafted BM-MSCs, we performed fibrosis and function
analysis. Representative Masson’s trichrome staining results
Fig. 8 Evaluation of fibrosis and heart function after myocardial infarction (MI). a Representative Masson’s trichrome staining revealed left
ventricular fibrosis 4 weeks after MI (magnification 4×). Quantitative analysis of the fibrotic area (b) and infarct wall thickness (c). Histograms
illustrating heart function parameters: left ventricular end-diastolic diameter (LVEDd, d), left ventricular end-systolic diameter (LVESd, e), left ventricular
ejection fraction (f) and left ventricular fractional shortening (g). Data are expressed as means ± SEM; n = 5; *p < 0.05. 3-MA 3-methyladenine, HPCMSCs
hypoxic preconditioned mesenchymal stem cells, LV left ventricle, NPCMSCs normal preconditioned mesenchymal stem cells, Sham sham-operated,
siRNA small interfering RNA
showed that HPCBM-MSCs decreased fibrosis after MI
(Fig. 8a and b). Moreover, infarct wall thickness (Fig. 8c) in
the hearts from mice transplanted with hypoxic
preconditioned BM-MSCs was increased compared to that from
mice treated with normoxia cultured BM-MSCs. Serial
echocardiographic analysis indicated that the baseline
parameters were similar in all groups. The LV dimensions
(LVEDD and LVESD) were increased after MI. Meanwhile,
the LV dimensions were decreased in the HPCBM-MSC
group compared with the MI and NPCBM-MSC groups
(Fig. 8d and e). Moreover, transplantation of HPCBM-MSCs
also manifested a trend towards improvement of cardiac
performance over the 4 weeks after MI. However, the
apparent benefit of HPCBM-MSC transplantation was
abolished by autophagy inhibition with 3-MA and Atg7
siRNA(Fig. 8f and g). Collectively, these results suggest that
the therapeutic benefits of BM-MSC transplantation after
MI is enhanced by hypoxic preconditioning via an
In the present study, we demonstrate for the first time
that hypoxic preconditioning exerts a protective effect
on BM-MSCs against hypoxia and nutrient deprivation
in vitro, associated with autophagy regulation. In
addition, hypoxic preconditioning also increases the
survival of BM-MSCs after transplantation and further
enhances their therapeutic potential for MI in vivo.
Furthermore, the beneficial effects generated from the
hypoxia preconditioning of BM-MSCs are mediated in
an autophagy-dependent manner. Overall, this study
demonstrates that hypoxic preconditioning may be a
potential optimizing target for BM-MSC-based cellular
therapy for MI.
Stem cell-based therapy for MI has been considered
as a potential strategy for MI. However, previous
studies revealed that the therapeutic benefits seem to be
relatively modest . Meanwhile, our clinical trials also
revealed that intracoronary administration of
autologous bone marrow mononuclear cells (BMMNC) can
lead to no significant myocardial functional
improvement after 4 years of follow-up . Poor viability and
low cell retention of donor cells in the ischemic
myocardium are thought to be a primary limitation for the
clinical application of cell therapy. Mangi et al.  and
van der Bogt et al.  found that autologous
BMMSCs had undergone acute death within 1 week after
transplantation and the survival rate of engrafted cells
was only 1% in the ischemic heart. Consistently, our
previous studies also demonstrated a massive cell death
between days 3 and 7 after transplantation [7, 18]. In
the present study, we performed in vivo BLI assay to
track the engrafted BM-MSCs, which were isolated
from reporter transgenic mice and constitutively
express Fluc and GFP . The BLI results showed
acute cell death within 7 days after engraftment into
the infarcted myocardium. Moreover, BM-MSC
transplantation could not improve the cardiac function
significantly, which was consistent with previous studies
. Furthermore, the histological assays revealed that
GFP+ cells were increased in the HPCMSC group,
indicating that HPC enhanced the survival of BM-MSCs
after transplantation. In addition, the
immunofluorescent staining for Ki-67 and TUNEL also revealed that
HPC increased the viability of transplanted MSCs with
decreased apoptosis. Taken together, these data
suggested that HPC enhanced the survival and
proliferation of engrafted cells, which contributed to prolonged
duration of BM-MSCs in the infarcted heart.
Although the cause of transplanted cell death remains
to be elucidated, the noxious milieu in the ischemic
myocardium, coupled with enhanced inflammation,
oxidative stress, and accumulation of cytotoxic substances,
offers a significant challenge to the transplanted donor
stem cells. Therefore, strategies aimed at improving the
adaptation of transplanted MSCs to the harsh hypoxic
conditions are crucial for improving the efficiency of cell
therapy. Many methods, including gene modification
 and pharmaceutical approaches [7, 18], have been
suggested to be effective in reinforcing the viability of
the donor cells. However, these approaches are not
suitable for the clinic because of some potential drawbacks,
including insertional mutagenesis, high manufacturing
costs, and being time consuming. Therefore, simpler and
safer methods to enhance the therapeutic efficacy need
to be explored.
Preconditioning of donor cells has recently attracted
attention as an optimized strategy to increase the
therapeutic efficacy of stem cell-based treatment of ischemic
diseases [24, 25]. Although MSCs normally reside in a
physiologically hypoxic niche, such as bone marrow
and adipose tissue, the ex vivo culture condition is
normoxic [26, 27]. Once transplanted into the ischemic
myocardium, MSCs encounter severe hypoxia and a
cytokine-rich microenvironment, which results in
extensive apoptosis. However, sublethal hypoxic culture
(1%–3% O2) before exposure to the severe ischemia has
been proved to be beneficial for MSCs, as this oxygen
tension is more similar to the physiologic niche of
MSCs. Previous studies demonstrated that hypoxic
preconditioning increased the survival and therapeutic
potency of stem cells [9, 26, 28–30]. However, the
detailed preconditioning protocols used in previous
studies varied widely. In the present study, we found
that the beneficial effects of HPC with 1% O2 for 24 h
are most pronounced, which was in line with most
HPC protocols described for human or animal bone
marrow MSCs. Long-term ischemic preconditioning,
however, proved to be detrimental to BM-MSCs.
Therefore, the optimized HPC protocol (with both 1%
O2 for 24 h and 48 h) was used in our study.
Previous studies have demonstrated that HPC can
increase the therapeutic effects of transplanted
stem/progenitor cells in ischemic diseases, such as in the limb
, cerebral , renal , and spinal cord . To
intuitively reveal the effect of HPC on the survival of
BM-MSCs in the ischemic heart, we performed the in
vivo BLI assay and found the ameliorated survival of
HPCMSCs compared with NPCMSCs. Moreover,
transplantation with HPCMSCs was effective at preventing
cardiomyocyte apoptosis, decreasing myocardial
fibrosis, increasing the number of new capillaries, and
improving cardiac function. Taken together, our results
indicated that HPC for 24 h promoted the viability of
BM-MSCs in the infarcted heart and even enhanced
the therapeutic efficiency for MI, while long-term HPC
promoted apoptosis and the mortality rate of
BMMSCs in the infarcted heart.
The mechanisms underlying the beneficial effects of
hypoxia preconditioning on MSCs remain incompletely
understood. Autophagy is a tightly regulated catabolic
mechanism that is essential to maintain homeostasis and
normal physiological functioning . It has been proved
that various stressful conditions, including hypoxia and
nutrition deprivation, can induce autophagy .
However, the relationship between autophagy and cell death is
controversial. Moreover, the detailed effect of HPC on
autophagy of MSCs has not been fully understood.
Therefore, we hypothesized that the protective effects
of HPC on BM-MSCs are mediated by regulating
autophagy. In the present study, hypoxic preconditioning
(1% oxygen) caused time-dependent autophagy, which
was demonstrated by an increase in LC3 expression
and the formation of autophagic vacuoles (Fig. 4).
However, HPC for 36 h or 48 h was detrimental to
BMMSCs in vitro, indicating that dramatically upregulated
autophagy induced by long-term HPC may reversely
lead to programmed cell death. Furthermore, 3-MA, an
autophagic inhibitor that inhibits class III PI3K,
blocked the HPC-mediated protection. Consistently,
inhibition of the autophagic initiation by Atg7 siRNA also
eliminated the beneficial effect of HPC. Moreover, the
therapeutic effects of HPC-MSC transplantation were
also abolished by autophagy inhibition with 3-MA or
Atg7 siRNA. Taken together, these results confirm that
modest HPC-induced protection (with 1% O2 for 24 h) is
likely mediated by regulating the autophagic pathway.
There is still substantial controversy on the detailed
mechanisms of stem cell therapy for ischemic diseases.
Although transdifferentiation into cardiomyocytes in
cardiac niches was proposed as the key mechanism, a
growing body of evidence suggests that paracrine
mechanisms mediated by MSCs may play a more
essential role . Our previous study revealed that
BMMSCs secreted all kinds of bioactive factors, such as
VEGF, bFGF, HGF, and IGF-1. In the present study, we
found that the paracrine secretion effects of BM-MSCs
were enhanced by our optimized HPC protocol (with
1% O2 for 24 h). Moreover, VEGF, bFGF, HGF, and
IGF-1 were downregulated with HPC for 48 h.
However, those secretions were dramatically elevated after
autophagy inhibition (by 3-MA or Atg7 siRNA
administration). Therefore, appropriate autophagy induced by
HPC for 24 h could significantly promote paracrine
secretion. Furthermore, transplantation of HPCMSCs
significantly increased the number of capillaries in the
peri-infarct zone, indicating that HPC enhanced the
angiogenic potential of BM-MSCs. However, both
apoptosis and autophagy levels were significantly increased
with HPC for 48 h. Furthermore, the apoptosis was
decreased, while paracrine secretions were dramatically
elevated, after autophagy inhibition by 3-MA and Atg7
siRNA. Therefore, excessive autophagy can increase the
apoptosis level thereby affecting BM-MSC function. Taken
together, our results suggest that autophagy plays a
double-edged sword under different HPC protocols.
In conclusion, the current study confirmed that HPC for
24 h has the potential to enhance the functional survival
of MSCs implanted into the infarcted heart. In addition,
MSCs with HPC for 24 h before transplantation exhibited
a significant improvement in cardiac function. The
favorable effect of HPC on MSCs could be attributed to
regulation of autophagy. Therefore, our results suggest that
appropriate hypoxic preconditioning may be a promising
novel approach for optimizing MSC transplantation
therapy for MI.
Additional file 1: Figure S1. The effect of autophagy inhibition on the
paracrine secretions of BM-MSCs treated by HPC for 24 h. ELISA assay
demonstrated the levels of VEGF (A), bFGF (B), IGF-1 (C), and HGF (D)
secreted by BM-MSCs treated by HPC for 24 h with and without autophagy
inhibition by 3-MA and Atg7 siRNA. Data are expressed as means ± SEM;
n = 5; *p < 0.05. (TIF 626 kb)
Additional file 2: Figure S2. Effect of HPC for 48 h on the paracrine
secretion of BM-MSCs. ELISA assay demonstrated the levels of VEGF (A),
bFGF (B), IGF-1 (C), and HGF (D) secreted by BM-MSCs treated by HPC for
48 h with and without autophagy inhibition by 3-MA and Atg7 siRNA.
Data are expressed as means ± SEM; n = 5; *p < 0.05. (TIF 663 kb)
Additional file 3: Figure S3. Autophagy modulated the adverse effect
of HPC for 48 h on BM-MSCs. (A) Representative TUNEL images of BM-MSCs
treated by HPC for 48 h with or without autophagy inhibition by 3-MA and
Atg7siRNA. Scale bars = 20 μm. (B) The quantification of the apoptotic
BMMSCs in all groups. Data are expressed as means ± SEM; n = 5; *p < 0.05.
(TIF 2401 kb)
Additional file 4: Figure S4. Effect of 3-MA and Atg7 siRNA on the
autophagy of BM-MSCs with HPC. (A) Representative immunofluorescence
images of GFP-LC3 (green fluorescence) and DAPI (blue fluorescence) in
BM-MSCs with HPC and the autophagy inhibitor 3-MA and Atg7 siRNA,
respectively. Scale bars = 20 μm. (B) Quantification of autophagy flux was
presented as the percentage of BM-MSCs with punctate LC3 in all groups.
*p < 0.05. (TIF 1978 kb)
Additional file 5 Figure S5. Evaluation of the survival of transplanted
BM-MSCs with different HPC protocols. (A) Representative longitudinal BLI
spatiotemporally tracked BM-MSCs (top row, n = 10), HPC 24 h MSCs
(second row, n = 10), and HPC 48 h MSCs (third row, n = 10). Color scale
bar values are in photons/s/cm2/sr. (B) Quantitative analysis of Fluc
optical signals on fixed regions of interest (ROI). (TIF 3197 kb)
αMEM: Alpha-modified Eagle’s medium; 3MA: 3-Methyladenine;
ANOVA: Analysis of variance; ATG: Autophagy associated gene; bFGF: Basic
fibroblast growth factor; BLI: Bioluminescence imaging; BM-MSC: Bone
marrow mesenchymal stem cell; DAPI: 4,6-Diamidino-2-phenylindole;
DMEM: Dulbecco’s modified Eagle’s medium; eGFP: Enhanced green
fluorescent protein; ELISA: Enzyme-linked immunosorbent assay; FBS: Fetal
bovine serum; FCS: Fetal calf serum; Fluc: Firefly luciferase; FS: Fractional
shortening; H/SD: Hypoxia/serum deprivation; HGF: Hepatocyte growth
factor; HPC: Hypoxic preconditioning; HPCMSC: Hypoxic preconditioned (bone
marrow) mesenchymal stem cell; IGF: Insulin-like growth factor; LAD: Left
anterior descending; LVEDD: Left ventricular end-diastolic diameter;
LVEDV: Left ventricular end-diastolic volume; LVEF: Left ventricular ejection
fraction; LVESD: Left ventricular end-systolic diameter; LVESV: Left ventricular
end-systolic volume; MI: Myocardial infarction; MTT:
3-(4,5-Dimethylthiazol-2yl)-2,5- diphenyltetrazolium bromide; NPCMSC: Normal preconditioned (bone
marrow) mesenchymal stem cell; OD: Optical density; PBS:
Phosphatebuffered saline; siRNA: Small interfering RNA; TUNEL: Terminal
deoxynucleotidyl transferase-mediated nick-end labeling; VEGF: Vascular
endothelial growth factor
ZZ, CY, MS, HC, and MY designed the study, drafted the manuscript, and
approved its final version. TH acquired data, revised the article’s intellectual
content, and approved the final version. ZJ, LD, WJ, and JY are responsible for
the integrity of this work. All authors read and approved the final manuscript.
All procedures were performed in accordance with the institutional guidelines for
animal research and were approved by the Animal Care and Use Committee of
the Second Artillery General Hospital of PLA.
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