Mesenchymal stromal cells ameliorate oxidative stress-induced islet endothelium apoptosis and functional impairment via Wnt4-β-catenin signaling
Wang et al. Stem Cell Research & Therapy
Mesenchymal stromal cells ameliorate oxidative stress-induced islet endothelium apoptosis and functional impairment via Wnt4-β-catenin signaling
Lingshu Wang 0
Li Qing 0
He Liu 0
Na Liu 2
Jingting Qiao 0
Chen Cui 0
Tianyi He 0
Ruxing Zhao 0
Fuqiang Liu 0
Fei Yan 0
Chuan Wang 0
Kai Liang 0
Xinghong Guo 0
Ying H. Shen 1 3
Xinguo Hou 0
Li Chen 0
0 Department of Endocrinology, Institute of Endocrinology and Metabolism, Qilu Hospital of Shandong University , Jinan 250012, Shandong , China
1 Division of Cardiothoracic Surgery, Michael E. DeBakey Department of Surgery, Baylor College of Medicine , Houston, TX , USA
2 College of Public Health, Shandong University , Jinan, Shandong 250012 , China
3 Texas Heart Institute , Houston, TX , USA
Background: Islet dysfunction and destruction are the common cause for both type 1 and type 2 diabetes mellitus (T2DM). The islets of Langerhans are highly vascularized miniorgans, and preserving the structural integrity and full function of the microvascular endothelium is vital for protecting the islets from the infiltration of immune cells and secondary inflammatory attack. Mesenchymal stromal cell (MSC)-based therapies have been proven to promote angiogenesis of the islets; however, the underlying mechanism for the protective role of MSCs in the islet endothelium is still vague. Methods: In this study, we used MS-1, a murine islet microvascular endothelium cell line, and an MSC-MS1 transwell culturing system to investigate the protective mechanism of rat bone marrow-derived MSCs under oxidative stress in vitro. Cell apoptosis was detected by TUNEL staining, annexin V/PI flow cytometry analysis, and cleaved caspase 3 western blotting analysis. Endothelial cell activation was determined by expression of intercellular cell adhesion molecule (ICAM) and vascular cell adhesion molecule (VCAM), as well as eNOS phosphorylation/ activation. The changes of VCAM-1, eNOS, and the β-catenin expression were also tested in the isolated islets of T2DM rats infused with MSCs. Results: We observed that treating MS-1 cells with H2O2 triggered significant apoptosis, induction of VCAM expression, and reduction of eNOS phosphorylation. Importantly, coculturing MS-1 cells with MSCs prevented oxidative stressinduced apoptosis, eNOS inhibition, and VCAM elevation in MS-1 cells. Similar changes in VCAM-1 and eNOS phosphorylation could also be observed in the islets isolated from T2DM rats infused with MSCs. Moreover, MSCs cocultured with MS-1 in vitro or their administration in vivo could both result in an increase of β-catenin, which suggested activation of the β-catenin-dependent Wnt signaling pathway. In MS-1 cells, activation of the β-catenindependent Wnt signaling pathway partially mediated the protective effects of MSCs against H2O2-induced apoptosis and eNOS inhibition. Furthermore, MSCs produced a significant amount of Wnt4 and Wnt5a. Although both Wnt4 and Wnt5a participated in the interaction between MSCs and MS-1 cells, Wnt4 exhibited a protective role while Wnt5a seemed to show a destructive role in MS-1 cells. Conclusions: Our observations provide evidence that the orchestration of the MSC-secreted Wnts could promote the survival and improve the endothelial function of the injured islet endothelium via activating the β-catenin-dependent Wnt signaling in target endothelial cells. This finding might inspire further in-vivo studies.
Islet endothelium; Mesenchymal stromal cell; Wnt
Type 2 diabetes mellitus (T2DM) has become a global
health issue due to its increasing morbidity and
aggravating financial burden. According to ADA reports, the
prevalence of T2DM in the USA has reached a plateau at
approximately 12% [
]; however, the prevalence of
diabetes and prediabetes is still increasing in China and has
reached up to 11.6% and 50.1%, respectively, in the adult
population of China [
]. With the development of T2DM,
insulin resistance is present throughout the disease
process; islet function exhibits a compensatory elevation
in the early stage and undergoes a constant decline
afterward. Thus, preserving islet function is the key in
managing glucose and preventing T2DM progression.
The islets are highly vascularized miniorgans, with
their combined 1–2% of the pancreatic volume receiving
10–20% of the total pancreatic blood flow [
of this unique structure, the islets are able to respond
rapidly to glucose and hormone fluctuations but are also
vulnerable to unfavorable stimuli such as oxidative
stress. Once the microvascular integrity of the islets is
impaired, together with the disturbance of the
NOmediated endothelium vasodilation [
] and the
upregulation of adhesion molecules [
], it becomes easier for the
inflammatory cells to adhere and migrate into the islet
and cause further islet destruction [
]. Considering the
importance of islet microvascular endothelium,
preserving its integrity and proper function might be a novel
target in islet protection.
Mesenchymal stromal cell (MSC)-based therapies have
been proven effective in various clinical trials for both
T1DM and T2DM [
], as well as in diet-induced [
or genetically modified experimental [
animal models. Systemic MSC infusion alone has been shown
to alleviate hyperglycemia, improve islet function, and
attenuate insulin resistance, while MSC and islet
cotransplantation facilitates graft revascularization and promotes
graft survival [
]. Relatively few studies have focused
on changes in the existing islet microvascular endothelium
after MSC transplantation , and the aforementioned
studies analyzed the change in endothelial cell numbers
but not the function of the islet vasculature, partially due
to the difficulties of measuring the islet blood flow in vivo.
Furthermore, the mechanism for the protective role of
MSCs in islet endothelium is still vague. Considering the
pro-angiogenesis nature of MSCs and their diverse
], it is reasonable to assume that MSCs could alter
endothelial cell behavior and protect the islet endothelium
from destructive stimuli by secreting various factors.
Therefore, in this study we used MS-1, a murine islet
microvascular endothelium cell line, and rat primary
bone marrow-derived MSCs to investigate the secreted
factors and downstream pathways responsible for the
protective effects of MSCs. To simulate a paracrine
microenvironment, we built an MSC-MS1 transwell
culturing system to investigate the protective mechanism of
MSCs under oxidative stress, and the results might
inspire further in-vivo studies.
Cell culture and treatments
Rat primary bone marrow MSCs (bmMSCs) were
obtained by isolating the femurs of rats, flushing the
marrow, and cultivating the eluent in Dulbecco’s modified
Eagle’s medium (low glucose, 5.5 mmol/l (L-DMEM))
supplemented with 20% fetal bovine serum (FBS). After
24 h, the supernatant was discarded, and the culturing
was continued until the cells reached 80% confluency.
After the first passage, the MSCs were cultured in
LDMEM with 10% FBS. The third passage was used for
conditioned medium (CM) gathering, differentiation
induction, flow cytometry analysis for surface markers,
and transwell culturing. The MSCs were cultured in the
upper chamber of the transwell system (catalog no.
3414; Corning, USA).
The primary lung epithelial cells were obtained from
newborn rats. The rats were sacrificed, and the lungs
were separated, cut into small pieces, and digested in
0.25% trypsin. The cell suspension was cultivated in
LDMEM plus 10% FBS for 24 h, and the nonadherent
cells were discarded.
MS-1 cells (MILE SVEN 1, ATCC Number: CRL-2279™)
were cultured in H-DMEM supplemented with 5% FBS
according to the supplier’s protocol (https://www.atcc.org/
were given after 24 h of serum starvation when the cells
reached 60% confluency. For dose-dependent effects,
MS1 cells were treated with 0, 50, 100, 200, 400, and
800 μmol/L H2O2 for 24 or 48 h, and cell viability was
detected using MTT. The optimal H2O2 concentration was
that under which the cell viability dropped to 50–60%
compared to cell viability of the control. The
concentration of H2O2 used for the rest of the experiments was
200 μmol/L according to the results. The
pharmacological inhibitor XAV-939 (catalog no. S1180;
Selleckchem, USA), an inhibitor of β-catenin transcriptional
activity, was incubated together with the other
treatments to block the canonical Wnt signalling. The
optimized concentration of XAV-939 was confirmed by
treating MS-1 cells with 0, 2.5, 5, 10, 20, and 40 μmol/L
XAV-939 for 24 h and extracting the proteins for
western blotting analysis. The proper concentration was the
minimal concentration that significantly reduced the
amount of β-catenin while not increasing the
proportion of cleaved caspase 3. Finally, 10 μmol/L of
XAV939 was used in the following experiments, and this
concentration was consistent with that used in previous
Cell viability and apoptosis
Cell viability was detected using the MTT method.
MS1 cells were seeded in 96-well culture plates at 1 × 105
cells/well. Four hours before analysis, 5 mg/ml MTT
solution was added. For analysis, the supernatant was
removed, and cells were solubilized in acid isopropyl
alcohol. The absorption was measured by
spectrophotometry at 570 nm with reference at 630 nm.
Cell apoptosis was detected using an In Situ Cell Death
Detection kit (catalog no. 12156792910; Sigma-Aldrich,
USA), FITC Annexin V Apoptosis Detection kit (catalog
no. 556547; BD Pharmingen, USA), and western blotting
analysis for the percentage of cleaved caspase 3 and 7.
In-situ cell death detection (TUNEL) was conducted
following the manufacturer’s protocol. In brief, cells
were planted on coverslips in six-well plates. After
treatment, slides were washed three times with ice-cold PBS
and fixed with 4% paraformaldehyde. The cells were
incubated with 1% Triton X-100 for 5 min and then
incubated at 37 °C with 50 μl/slide TUNEL reaction mixture
in darkness for 60 min. After incubation, the slides were
washed three times and stained with Hoechst33258 for
5 min. Apoptotic cells were counted in random fields by
fluorescence microscopy; each experiment was
performed in triplicate (×40 magnification, at least 10 fields
FITC annexin V apoptosis detection was performed
using flow cytometry. Cells were planted on six-well
plates. After treatment, cells were stained with annexin
V for 20 min at room temperature in the dark, and
propidium iodide (PI) was added 5 min before analysis.
The nuclear transposition of β-catenin in MS-1 cells was
detected by indirect immunofluorescence. The slides
were prepared as already described. After incubation in
1% Triton X-100 for 5 min, the cells were incubated
with 1:500 rabbit anti-rat β-catenin antibody (catalog no.
ab32572; Abcam, USA) in 1% BSA in PBS overnight at
4 °C. The slides were incubated with goat anti-rabbit
Alexa Fluor 488 (catalog no. 1515529; Life Technology,
USA) diluted 1:500 in 1% BSA in PBS for 60 min and
then DAPI for 10 min. The transposition of β-catenin
was observed under laser scanning confocal microscopy.
Real-time quantitative PCR
The total mRNA was extracted using an EZNA
MicroElute Total RNA Kit (catalog no. R6831-01; Omega
BioTek, USA) under the manufacturer’s instructions and
then reverse-transcribed using a Prime Script RT
Reagent Kit (catalog no. RR047A; Takara, Japan). Primers
were designed with Primer Premier 6.0 software and
synthesized chemically by Sangon Biotech (Shanghai)
Co., Ltd. The primers were as follows: Wnt2, sense
5′CTCGGTGGAATCTGGCTCTG-3′ and antisense 5′-C
ACATTGTCACACATCACCCT-3′; Wnt3a, sense 5′-G
TTTGCCGATGCCAGGGAGAA-3′ and antisense 5′-A
CCACCAGCAGGTCTTCACTTC-3′; Wnt4, sense 5′-A
GACGTGCGAGAAACTCAAAG-3′ and antisense 5′-G
GAACTGGTATTGGCACTCCT-3′; Wnt5a, sense 5′-G
CAGGTCAACAGCCGCTTCAACTC-3′ and antisense
5′-TCATAGCCACGCCCACAGCACAT-3′; and Wnt10b,
sense 5′-GGACGCCAGGTGGTAACGGAAA-3′ and
Realtime PCR was conducted with the SYBR Green PCR kit
(catalog no. RR820B; Takara), and quantification was
achieved by normalization using β-actin as the control.
Western blotting analysis
Whole-cell proteins were extracted by RIPA lysis buffer
(catalog no. P0013B; Biotime, China). The plasma
proteins were extracted by Tris-Triton (10 mM Tris,
100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton
X-100, 10% glycerol, 0.1% SDS, and 0.5% deoxycholate),
and the nuclear proteins were extracted by RIPA.
Proteins were separated by 10% SDS-PAGE and transferred
to nitrocellulose membranes. The membranes were
blocked in 5% nonfat milk in TBS-T (50 mmol/l Tris,
pH 7.5, 150 mmol/l NaCl, 0.05% Tween 20) for 1 h at
room temperature and incubated in primary antibodies
at 4 °C overnight. Bound primary antibodies were
detected by horseradish peroxidase-conjugated secondary
antibodies for 1 h at room temperature and visualized by
enhanced chemiluminescence (Amersham Imager 600,
GE, USA). Quantification of bands were performed
using ImageJ software.
The primary antibodies were as follows: β-catenin,
cyclin D1 (catalog no. ab134175; Abcam), Histone H3
(catalog no. 4499P; Cell Signaling Technology, USA), p-eNOS
(catalog no. ab184154; Abcam), total-eNOS (catalog no.
ab66127; Abcam), ICAM (catalog no. BA0541; Boster,
China), VCAM (catalog no. XBA0406; Boster), Wnt4
(catalog no. sc376279; Santa Cruz, USA), Wnt5a (catalog
no. sc365370; Santa Cruz), and β-actin (catalog no.
BM0627; Boster), while cleaved/total caspase 3/7 were
from an Apoptosis Antibody Sampler Kit (catalog no.
9915; Cell Signaling Technology, USA).
Silencing RNA knockdown
Silencing RNA (siRNA) oligonucleotides were synthesized
by Shanghai GenePharma Co., Ltd. The sequences of
negative control (NC) siRNA were sense 5′-UCCUCCG
AACGUGUCACGUTT-3′ and antisense 5′-ACGUGAC
ACGUUCGGAGAATT-3′. The sequences for the Wnt4
siRNA were sense 5′-GCCAAGUCCAGACUUCUGUT
T-3′ and antisense 5′-ACAGAAGUCUGGACUUGGC
TT-3. The sequences for the Wnt5a siRNA were sense
5′GAAGCCCAUUGGAAUAUUATT-3′ and antisense
5′UAAUAUUCCAAUGGGCUUCTT-3′. The siRNAs for
Wnt4 and Wnt5a could suppress the corresponding
mRNA levels to less than 10% compared to that expressed
in NC siRNAs, and the samples were also tested for other
Wnt mRNAs to guarantee that the knockdown was
specific and no off-target effects occurred (data not shown).
The siRNAs were transfected into rat MSCs using the
Lipofectamine RNAiMAX Transfection Reagent (catalog
no. 13778100; Invitrogen, USA) according to the
manufacturer’s instructions. In brief, the MSCs were seeded in
transwell chambers or in culture flasks until the cells
reached 80–90% confluency. The medium was then
removed and replaced with Opti-MEM I Reduced Serum
Medium (catalog no. 31985070; Invitrogen), in which 1 ×
106 cells were subjected to 25 pmol siRNA mixed with
7.5 μl transfection reagent. After a 48-h incubation, the
supernatant was discarded and was replaced with
LDMEM with 10% FBS. The efficiency of RNA knockdown
was evaluated by qPCR and western blotting analysis. The
cells in the transwell chamber were used for transwell
culturing, and the supernatant was gathered as CM.
Animals and bmMSC infusion
The T2DM rat model was established by a continuously
high-fat diet (HFD) combined with a single dose of STZ
(30 mg/kg, catalog no. S0130; Sigma-Aldrich) at the
fourth week of HFD. Diabetes was identified as fasting
glucose ≥ 16.7 mmol/L twice in succession. Then 5 × 106
cells/rat of primary bmMSCs at passage 3 were
administered intravenously 7 days after the STZ injection. The
untreated T2DM rats were infused with physiological
saline. Fasting blood glucose was monitored weekly by
Accu-Chek® Performa (Roche Life Science, USA).
Intraperitoneal glucose tolerance test
The intraperitoneal glucose tolerance tests (IPGTTs)
were performed 2, 4, and 8 weeks after the MSC
infusion. After overnight fasting, the rats were anesthetized
with isoflurane and injected intraperitoneally with 1.5 g/
kg glucose. Blood was collected from the tail vein before
(0 h) and 0.5, 1, 2, and 3 h after the glucose injection for
glucose and insulin testing. Glucose was measured by
Accu-Chek® Performa, and serum insulin was measured
by radioimmunoassay in the Department of Nuclides,
Isolation, purification, and protein extraction of islets from rats
Rat islets were isolated from the nondiabetic (NDM)
control rats, the nontreated T2DM rats, and the
bmMSCinfused T2DM rats (N = 3, respectively). The islets were
isolated by collagenase digestion (1 mg/ml, type V, catalog
no. C9263; Sigma-Aldrich) followed by hand picking
under a stereoscopic microscope (Olympus SZX7). In
brief, we first separated the common bile duct and ligated
it close to the duodenum. A 4.5-sized needle was placed
into the engorging bile duct and the backflow of the bile
could be observed. We then sacrificed the rat, waited until
the duodenum turned pale, clipped the porta hepatis, and
injected 6 ml of ice-cold collagenase V (1 mg/ml) slowly
into the bile duct to let the pancreas swell. The pancreas
was isolated and incubated in 5 ml Hanks solution under
38 °C for 13 min. The pancreas was then shaken with mild
wrist force until it disintegrated into a fine sand-like
suspension, 20 ml of ice-cold Hanks + 10% bovine serum was
added to stop the digestion, and the suspension was passed
through a 30-mesh screen. The sample was centrifuged at
1000 rpm for 1 min, then we discarded the supernatant,
and washed the sedimentation with ice-cold Hanks
solution for three times. To purify the isolated islets, we picked
the islets under the stereoscopic microscope. The isolated
islets were identified by dithizone (DTZ) staining and
about 300 islets could be obtained from a single rat. These
islets were lysed in 200 μl RIPA, and the protein extraction
procedure was as already described.
Differences between two groups were evaluated using a
Student’s t test and the χ2 test; for three groups or more,
a one-way ANOVA was used. p < 0.05 was considered
statistically significant. All of the statistical analyses were
performed with SPSS 16.0 software.
Coculturing bmMSCs and MS-1 cells ameliorated H2O2-in
duced apoptosis and functional impairment
The bone marrow-derived MSCs (bmMSCs) were
identified by the expression of multiple surface markers and
differentiation toward osteoblasts and adipocytes. After
induction, the cells could differentiate toward osteoblasts
and adipocytes (Fig. 1a), which were identified by Van
Kossa silver stain for the calcium nodes (black) and
Oilred O stain for the lipid droplets (red). The MSCs were
positive for stem cell markers CD90 and CD44 and
negative for hematopoietic markers CD34 and CD45 (Fig. 1b).
After the identification of MSCs, we then tested the
effects of MSCs on oxidative stress-induced endothelium
injury. Oxidative stress-induced MS-1 cell injury was
established by exogenous administration of 200 μmol/L
H2O2 in cultured MS-1 cells. A significant decline in cell
viability was observed by MTT tests (Fig. 1c), and a
remarkable elevation in apoptosis was confirmed by
annexin V/PI double-staining flow cytometry (Fig. 1d),
TUNEL staining (Fig. 1e), and cleaved caspase 3 western
blotting (Fig. 1f ). Meanwhile, impairment of endothelial
function was also observed by the reduction of eNOS
phosphorylation and increased expression of adhesion
molecule VCAM (Fig. 1f ).
However, when MS-1 cells were cultured with
MSCs in a transwell coculturing chamber,
H2O2-induced apoptosis declined dramatically, confirmed by
both TUNEL staining (Fig. 1e) and annexin V/PI flow
cytometry (Fig. 1d). The culture medium (CM) from
the MSCs also reversed the H2O2-induced reduction
in cell viability (Fig. 1c) and endothelial nitric oxide
synthase (eNOS) phosphorylation, as well as
H2O2-induced caspase3 cleavage/activation and vascular cell
adhesion molecule (VCAM) expression, suggesting
that MSCs could ameliorate oxidative stress-induced
endothelial injury and dysfunction, probably through
their paracrine function (Fig. 1f ).
MSCs activated the β-catenin-dependent Wnt signaling
pathway in MS-1 cells
Wnt proteins are a group of soluble factors that are highly
expressed in less mature cells such as stem cells, and their
proper functioning is very important for cell self-renewal
and stemness maintenance. To explore the possible
mechanism for the ameliorative effects of MSCs in oxidative
stress-induced endothelial injury, we first analyzed the
difference in Wnt mRNA expression between the MSCs
and MS-1 cells. We observed a significant increase in the
expression of Wnt4 and Wnt5a among all of the Wnts
analyzed, including Wnt2, Wnt3a, Wnt4, Wnt5a, and
Wnt10b, in the MSCs compared to that of the MS-1 cells,
raising the possibility that the Wnt proteins might be
involved in the interaction between the two cells (Fig. 2a).
As the Wnt proteins were secreted into the
intracellular space, they would bind to the corresponding
receptors, such as the Frizzled proteins, and activate
the downstream Wnt signaling pathways. To
determine whether coculturing with MSCs activated the
βcatenin-dependent canonical Wnt signaling pathway
in MS-1 cells, we analyzed the nuclear translocation
of β-catenin in the MSC-treated endothelium. We
observed an increase in FITC-β-catenin fluorescence in
the MS-1 nucleus after coculturing with MSCs
(Fig. 2b). Similar results were shown for the increase
in nuclear protein levels of β-catenin in MS-1 cells
treated with MSC-CM, whereas the cytoplasmic
protein level of β-catenin remained unchanged. We also
analyzed the protein expression of cyclin D1, a
βcatenin target gene, and found a significant elevation
after MSC-CM administration (Fig. 2c). These results
suggest that MSCs activated the β-catenin-dependent
Wnt signaling pathway in MS-1 cells.
The beneficial effects of MSCs were partially dependent
on the activation of the β-catenin-dependent Wnt
To examine whether MSCs act through activating the
βcatenin-dependent Wnt signaling pathway in MS-1 cells,
we used XAV-939, a Wnt/β-catenin-mediated
transcription antagonist that promotes the degradation of
βcatenin by stabilizing axin to block the
β-cateninassociated effects. XAV itself had little effect on MS-1,
but seemed to have a synergistic effect with H2O2 to
induce the apoptosis, eNOS phosphorylation impairment,
and VCAM expression upregulation of MS-1. After
blocking β-catenin, the protective role of MSCs against
H2O2 was significantly weakened, as shown by a decrease
in the cell viability (Fig. 3c), an increase in the apoptosis
rate (Fig. 3a, b), impaired eNOS phosphorylation, and
increased VCAM expression (Fig. 3d). However, MSCs did
actually ameliorate the H2O2 + XAV-939-induced cell
apoptosis to a certain extent, which suggested that the
beneficial effects of MSCs were conducted by the
activation of the β-catenin-dependent Wnt signaling pathway,
at least partially, in the islet microvascular endothelium.
MSC-secreted Wnt4 and Wnt5a were both involved in the response of MS-1 cells to oxidative stress but may have opposing effects
To demonstrate which Wnt protein was involved in the
protective process of MSCs toward endothelium injury,
we focused on the Wnts, whose expression differs the
most between MSCs and MS-1 cells. Therefore, we chose
Wnt4 and Wnt5a as possible candidates, and knocked
down their expression using siRNAs (Fig. 4a, b). Wnt4
had been proven to activate canonical Wnt signaling
pathways in cutaneous cells and artery endothelium, whereas
Wnt5a activated the noncanonical Wnt pathways. After
knocking down Wnt4 in the MSCs, the ameliorative
effects of MSCs were hampered (Fig. 4c–f ), which was
accompanied by a simultaneous decrease in the nuclear
translocation of β-catenin and downstream cyclin D1
expression (Fig. 5). However, Wnt5a knockdown seemed to
show the opposite effects: the protective effects were
reinforced, and the nuclear protein levels of cyclin D1 were
elevated, which suggested that MSC-secreted Wnt4 and
Wnt5a both influenced the response of MS-1 cells to
oxidative stress but may have opposing effects.
bmMSC infusion ameliorated hyperglycemia, improved
the islet β cell and endothelial function, and increased
the β-catenin nuclear translocation in high-fat diet and
STZ-induced T2DM rats
To evaluate the effects of MSCs on islet endothelium in
an in-vivo setting, we first built a T2DM rat model by
continuous HFD combined with a single dose of STZ
(30 mg/kg) at the fourth week of HFD. Then primary rat
bmMSCs were administered (5 × 106 cells/rat) 7 days
after the STZ injection. We found that the bmMSC
infusion significantly reduced the fasting glucose (Fig. 6a), as
well as improving the glucose tolerance during IPGTT at
the fourth week after MSC infusion (Fig. 6b). The
elevated insulin levels during IPGTT at week 4 were also
observed (Fig. 6c), so we chose week 4 as our
observation point in the following experiments.
Next we managed to evaluate the islet endothelial
function by quantifying the expression of p-eNOS and
VCAM. In order to rule out the disturbances of the
pancreatic endothelium, we isolated the islets from rats
4 weeks after the bmMSC infusion. The isolated islets
could be stained red by DTZ (Fig. 6d). Considering
eNOS was only expressed in the endothelium, and the
only endothelium in the isolated islet should be the islet
microvascular endothelium, we took total eNOS
(teNOS) as the internal reference to measure endothelial
p-eNOS and VCAM expression instead of β-actin. The
MSC administration significantly elevated the
phosphorylation of eNOS, and downregulated the expression of
VCAM, thus suggesting that bmMSC infusion could
improve the islet microvascular endothelium function in
vivo (Fig. 6e).
We also examined activation of the
β-catenindependent canonical Wnt signaling pathway in the islets.
The results of whole islet protein western blotting analysis
suggested a significant reduction of β-catenin in the islets
of T2DM rats, while MSCs infusion could improve the
βcatenin levels (Fig. 6e).
In this study, we investigated the possible mechanisms
by which MSCs prevent hydrogen peroxide-induced
injury in the MS-1 islet endothelium cell line; by secreting
Wnt4 and activating the β-catenin-dependent pathway
in MS-1 cells, MSCs could alleviate apoptosis and
improve endothelial function by promoting eNOS
phosphorylation and reducing the expression of adhesion
molecules such as VCAM.
Previous studies have suggested that systemic infusion
of MSCs could improve islet function and ameliorate
hyperglycemia, but studies focused on islet
microcirculation were rare and mostly focused on the impact of
cotransplanted MSCs on the islet graft. Rackham et al.
] reported that cotransplantation of MSCs could
increase the endothelial cell number in the graft, and Borg
et al.’s [
] study suggested that MSCs hastened the
revascularization of the transplanted islet cells, while
having no effects on the islet microvascular density. Cao
et al.’s [
] work pushed the knowledge of this topic a
step further; they suggested that MSCs could enhance
the peripheral vascular density of islet grafts by
differentiating into vascular smooth muscle cells and endothelial
cells and by secreting VEGF. Articles discussing the
impact of MSC transfusion on the existing islet
microvasculature were even scarcer; Bell et al. [
] transplanted a
group of selected MSCs with high-aldehyde
dehydrogenase activity into STZ-treated NOD/SCID
mice and observed elevated endogenous islet vascular
endothelium proliferation. However, due to the technical
difficulties of measuring islet blood flow in vivo by
microsphere measurements, hydrogen gas clearance, or
laser Doppler velocimetry [
], these studies only analyzed
the change in endothelium cell numbers but not the
function of the islet vasculature or the dilation capacity in
particular, which might be the main adaption in
hyperglycemia and insulin resistance. Although only an
in-vitro study, our study provided evidence that the
secreted factors of MSCs could improve the dilation
properties of the injured endothelium. By secreting
soluble Wnt proteins, MSCs could regulate the
activation of the β-catenin-dependent canonical Wnt
pathway in MS-1 cells, thus ameliorating oxidative
stressinduced cell apoptosis and preventing dilation failure
and proinflammatory adhesion molecule upregulation.
The Wnt protein family is a group of soluble proteins
that are secreted by less mature cells, such as stem cells
and tumors, and together with the activation of Wnt
pathways they participate in embryonic development,
stemness maintenance, and other pathological processes
such as neoplasia formation [
]. Normally, mature cells
express low levels of Wnt, but when the cells are under
stress Wnt expression might be activated from the
former quiescence state to take part in tissue repair [
Previous studies have established a link between the
Wnt signaling pathway and physiological/pathological
], but the results seemed to be
contradictory, which might be partially due to the different cell
types used and the different Wnt signaling pathways
activated. Although fewer studies have focused on the
relationship between Wnt signaling and islet function, the
existing findings appeared to be encouraging. Direct
deletion of β-catenin in the maturing beta cells disturbed
islet morphology and function, which led to severe
deregulation of glucose homeostasis [
]. TCF7L2, a vital
participant in the canonical Wnt signaling pathway, has
been proven to regulate glucose homeostasis by
preserving the beta cell mass [
]. Circulating Wnt
proteins have also been shown to be effective in regulating
islet function; Wnt3a activated canonical
β-catenindependent Wnt signaling to promote beta cell
proliferation both in vivo and in vitro [
], while Wnt-4
promoted beta cell proliferation but had no obvious impact
on secretion [
]. Although there were no articles
addressing the effect of Wnt signaling activation on the islet
endothelium, it is reasonable to assume that Wnts might
also participate in their response to injury.
Previous studies have reported that MSCs could
ameliorate tissue injury through secreting various Wnt
proteins in dermal cells and other cell types, but we were
the first to demonstrate that MSC-secreted Wnt could
improve eNOS phosphorylation/activation and reduce
VCAM expression in islet endothelial cells after
oxidative injury. A study by Zhang et al. [
that MSCs could accelerate the recovery of a cutaneous
burn by excreting Wnt4 packaged in exosomes, in which
Wnt4 also activated the canonical Wnt pathway and
induced the proliferation of dermal cells. In addition,
activation of the Wnt pathway by MSCs was also observed
in studies by Song et al. [
] and Leroux et al. [
showing beneficial effects on HCl-induced interstitial
cystitis and ischemia-induced muscle fiber injury.
In this study, we also observed an interesting
contradiction: MSCs simultaneously secreted “good” Wnts and
“bad” Wnts, but the final outcome seemed to be
beneficial. The Wnt proteins and the Wnt signaling pathway is
a complex network in which different Wnts bind to
different receptors, activating antagonistic pathways in a
dose-specific and cell-specific manner; the same Wnt
protein might activate opposing pathways in different
cells or by different concentrations [
]. Take Wnt4 as
an example; Wnt4 was discovered as a noncanonical
Wnt pathway activator but was later confirmed to be
able to act canonically through binding to LRP5 or LRP6
]. According to our observation, Wnt4 activated the
canonical Wnt pathway in the islet endothelium, which
was consistent with what has been reported in dermal
cells and muscle fibers [
]. Compared to Wnt4,
Wnt5a has been consistently believed to activate a
noncanonical Wnt signaling pathway and activates PKC or
JNK to conduct a proinflammatory effect [
our present study, when MSCs were under oxidative
stress, the expression of Wnt5a was significantly
elevated, whereas Wnt4 showed a downward trend. Their
effects were also observed to be in opposition, in line
with previous studies; MSC-secreted Wnt4 was proven
to take part in the canonical Wnt signaling activation
and partially mediated the protection of MSCs against
oxidative stress in islet microvascular endothelium,
whereas Wnt5a did the opposite. Fortunately, although
MSCs expressed an elevated level of Wnt5a under stress,
the anti-inflammation and anti-apoptotic effects were
still dominant. It is widely accepted that MSCs could be
induced by an inflammatory environment and polarized
into two subtypes: a proinflammatory subtype, MSC1;
and an anti-inflammatory subtype, MSC2 [
However, it is still not clear whether autocrine Wnts could
influence the immune polarization of MSCs. Based on
this hypothesis, one of our future directions is to
determine an intervention to antagonize the proinflammatory
environment-induced Wnt5a elevation and Wnt4
downregulation in MSCs in an attempt to minimize the
possibility of MSCs polarizing into a proinflammatory
subtype and to enhance their beneficial effects.
In summary, our observations provide evidence that the
orchestration of the MSC-secreted Wnts could promote
the survival and improve the endothelial function of the
injured islet endothelium. Our findings raise the
possibility that Wnt4 secreted by MSCs might improve the
islet endothelium function by activating the
β-catenindependent Wnt signaling pathway, but require further
CM: Conditioned medium; eNOS: Endothelial nitric oxide synthase; FBS: Fetal
bovine serum; ICAM: Intercellular cell adhesion molecule; MSC: Mesenchymal
stromal cell; NC: Negative control; PBS: Phosphate-buffered saline;
RTqPCR: Reverse transcription quantitative polymerase chain reaction;
siRNA: Silencing RNA; T2DM: Type 2 diabetes mellitus; VCAM: Vascular
cell adhesion molecule
The authors are grateful for the assistance of Professor Ping Yao at the
College of Public Health, Shandong University. This work could not be done
without them. The authors also want to thank American Journal Experts
(AJE) for English language editing.
This work was supported by the National Natural Science Foundation of China
(No. 81370943, No. 81670706, No. 81400769, No. 81500591, No. 81500592),
Shandong Provincial Department of Science and Technology—Innovation and
Achievement Transformation Special Grant (No. 2014ZZCX02201), the
International Science and Technology Cooperation Project Of Shandong
Province (No. 2010GHZ20201), and Qilu Research Foundation (No. 2015QLMS12).
Availability of data and materials
All data generated or analyzed during this study are included in this
LSW conceived this study, performed the experiments, collected data,
performed data analysis, and prepared the manuscript. LQ, HL, NL, JTQ, CC,
and TYH helped perform the experiments. RXZ, FQL, FY, CW, and KL helped
interpret the data and prepare the manuscript. XGH helped prepare the
manuscript. YHS, XGH, and LC conceived this study, performed data analysis,
and prepared and revised the manuscript. All authors read and approved the
The animal-related experiments, including the isolation of rat bone
marrow-derived mesenchymal stromal cells, the T2DM rat modeling, the
bmMSC infusion, and the isolation of rat islets, were approved by the
Animal Care and Utilization Committee of Shandong University (Ethics
Number DWLL-2015-005). All applicable institutional and national guidelines for
the care and use of animals were followed.
Consent for publication
All authors gave consent for publication.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published
maps and institutional affiliations.
Submit your next manuscript to BioMed Central
and we will help you at every step:
Our selector tool helps you to find the most relevant journal
1. Menke A , Casagrande S , Geiss L , Cowie CC . Prevalence of and trends in diabetes among adults in the United States, 1988 - 2012 . JAMA. 2015 ; 314 ( 10 ): 1021 - 9 .
2. Xu Y , Wang L , He J , Bi Y , Li M , Wang T , Wang L , Jiang Y , Dai M , Lu J , et al. Prevalence and control of diabetes in Chinese adults . JAMA . 2013 ; 310 ( 9 ): 948 - 59 .
3. Medarova Z , Castillo G , Dai G , Bolotin E , Bogdanov A , Moore A . Noninvasive magnetic resonance imaging of microvascular changes in type 1 diabetes . Diabetes . 2007 ; 56 ( 11 ): 2677 - 82 .
4. Garcia Soriano F , Virag L , Jagtap P , Szabo E , Mabley JG , Liaudet L , Marton A , Hoyt DG , Murthy KG , Salzman AL , et al. Diabetic endothelial dysfunction: the role of poly(ADP-ribose) polymerase activation . Nat Med . 2001 ; 7 ( 1 ): 108 - 13 .
5. Chakrabarti D , Huang X , Beck J , Henrich J , McFarland N , James RF , Stewart TA . Control of islet intercellular adhesion molecule-1 expression by interferonalpha and hypoxia . Diabetes . 1996 ; 45 ( 10 ): 1336 - 43 .
6. Olsson R , Carlsson PO . The pancreatic islet endothelial cell: emerging roles in islet function and disease . Int J Biochem Cell Biol . 2006 ; 38 ( 5-6 ): 710 - 4 .
7. Cai J , Wu Z , Xu X , Liao L , Chen J , Huang L , Wu W , Luo F , Wu C , Pugliese A , et al. Umbilical cord mesenchymal stromal cell with autologous bone marrow cell transplantation in established type 1 diabetes: a pilot randomized controlled open-label clinical study to assess safety and impact on insulin secretion . Diabetes Care . 2016 ; 39 ( 1 ): 149 - 57 .
8. Liu X , Zheng P , Wang X , Dai G , Cheng H , Zhang Z , Hua R , Niu X , Shi J , An Y. A preliminary evaluation of efficacy and safety of Wharton's jelly mesenchymal stem cell transplantation in patients with type 2 diabetes mellitus . Stem Cell Res Ther . 2014 ; 5 ( 2 ): 57 .
9. Si Y , Zhao Y , Hao H , Liu J , Guo Y , Mu Y , Shen J , Cheng Y , Fu X , Han W. Infusion of mesenchymal stem cells ameliorates hyperglycemia in type 2 diabetic rats: identification of a novel role in improving insulin sensitivity . Diabetes . 2012 ; 61 ( 6 ): 1616 - 25 .
10. Xie Z , Hao H , Tong C , Cheng Y , Liu J , Pang Y , Si Y , Guo Y , Zang L , Mu Y , et al. Human umbilical cord-derived mesenchymal stem cells elicit macrophages into an anti-inflammatory phenotype to alleviate insulin resistance in type 2 diabetic rats . Stem Cells (Dayton, Ohio) . 2016 ; 34 ( 3 ): 627 - 39 .
11. Ji AT , Chang YC , Fu YJ , Lee OK , Ho JH . Niche-dependent regulations of metabolic balance in high-fat diet-induced diabetic mice by mesenchymal stromal cells . Diabetes . 2015 ; 64 ( 3 ): 926 - 36 .
12. Jurewicz M , Yang S , Augello A , Godwin JG , Moore RF , Azzi J , Fiorina P , Atkinson M , Sayegh MH , Abdi R . Congenic mesenchymal stem cell therapy reverses hyperglycemia in experimental type 1 diabetes . Diabetes . 2010 ; 59 ( 12 ): 3139 - 47 .
13. Madec AM , Mallone R , Afonso G , Abou Mrad E , Mesnier A , Eljaafari A , Thivolet C . Mesenchymal stem cells protect NOD mice from diabetes by inducing regulatory T cells . Diabetologia . 2009 ; 52 ( 7 ): 1391 - 9 .
14. Johansson U , Rasmusson I , Niclou SP , Forslund N , Gustavsson L , Nilsson B , Korsgren O , Magnusson PU . Formation of composite endothelial cellmesenchymal stem cell islets: a novel approach to promote islet revascularization . Diabetes . 2008 ; 57 ( 9 ): 2393 - 401 .
15. Rackham CL , Vargas AE , Hawkes RG , Amisten S , Persaud SJ , Austin AL , King AJ , Jones PM . Annexin A1 is a key modulator of mesenchymal stromal cellmediated improvements in islet function . Diabetes . 2016 ; 65 ( 1 ): 129 - 39 .
16. Borg DJ , Weigelt M , Wilhelm C , Gerlach M , Bickle M , Speier S , Bonifacio E , Hommel A . Mesenchymal stromal cells improve transplanted islet survival and islet function in a syngeneic mouse model . Diabetologia . 2014 ; 57 ( 3 ): 522 - 31 .
17. Rackham CL , Chagastelles PC , Nardi NB , Hauge-Evans AC , Jones PM , King AJ . Co-transplantation of mesenchymal stem cells maintains islet organisation and morphology in mice . Diabetologia . 2011 ; 54 ( 5 ): 1127 - 35 .
18. Bell GI , Broughton HC , Levac KD , Allan DA , Xenocostas A , Hess DA . Transplanted human bone marrow progenitor subtypes stimulate endogenous islet regeneration and revascularization . Stem Cells Dev . 2012 ; 21 ( 1 ): 97 - 109 .
19. Bronckaers A , Hilkens P , Martens W , Gervois P , Ratajczak J , Struys T , Lambrichts I. Mesenchymal stem/stromal cells as a pharmacological and therapeutic approach to accelerate angiogenesis . Pharmacol Ther . 2014 ; 143 ( 2 ): 181 - 96 .
20. Cao XK , Li R , Sun W , Ge Y , Liu BL. Co-combination of islets with bone marrow mesenchymal stem cells promotes angiogenesis . Biomed Pharmacother . 2016 ; 78 : 156 - 64 .
21. Jansson L , Barbu A , Bodin B , Drott CJ , Espes D , Gao X , Grapensparr L , Kallskog O , Lau J , Liljeback H , et al. Pancreatic islet blood flow and its measurement . Ups J Med Sci . 2016 ; 121 ( 2 ): 81 - 95 .
22. Ring A , Kim YM , Kahn M. Wnt/catenin signaling in adult stem cell physiology and disease . Stem Cell Rev . 2014 ; 10 ( 4 ): 512 - 25 .
23. Krutzfeldt J , Stoffel M. Regulation of wingless-type MMTV integration site family (WNT) signalling in pancreatic islets from wild-type and obese mice . Diabetologia . 2010 ; 53 ( 1 ): 123 - 7 .
24. Dejana E. The role of wnt signaling in physiological and pathological angiogenesis . Circ Res . 2010 ; 107 ( 8 ): 943 - 52 .
25. Dabernat S , Secrest P , Peuchant E , Moreau-Gaudry F , Dubus P , Sarvetnick N. Lack of beta-catenin in early life induces abnormal glucose homeostasis in mice . Diabetologia . 2009 ; 52 ( 8 ): 1608 - 17 .
26. Takamoto I , Kubota N , Nakaya K , Kumagai K , Hashimoto S , Kubota T , Inoue M , Kajiwara E , Katsuyama H , Obata A , et al. TCF7L2 in mouse pancreatic beta cells plays a crucial role in glucose homeostasis by regulating beta cell mass . Diabetologia . 2014 ; 57 ( 3 ): 542 - 53 .
27. Yao DD , Yang L , Wang Y , Liu C , Wei YJ , Jia XB , Yin W , Shu L. Geniposide promotes beta-cell regeneration and survival through regulating betacatenin/TCF7L2 pathway . Cell Death Dis . 2015 ; 6 : e1746 .
28. Kozinski K , Jazurek M , Dobrzyn P , Janikiewicz J , Kolczynska K , Gajda A , Dobrzyn A . Adipose- and muscle-derived Wnts trigger pancreatic beta-cell adaptation to systemic insulin resistance . Sci Rep . 2016 ; 6 : 31553 .
29. Aly H , Rohatgi N , Marshall CA , Grossenheider TC , Miyoshi H , Stappenbeck TS , Matkovich SJ , McDaniel ML . A novel strategy to increase the proliferative potential of adult human beta-cells while maintaining their differentiated phenotype . PLoS One . 2013 ; 8 ( 6 ): e66131 .
30. Heller C , Kuhn MC , Mulders-Opgenoorth B , Schott M , Willenberg HS , Scherbaum WA , Schinner S . Exendin-4 upregulates the expression of Wnt-4, a novel regulator of pancreatic beta-cell proliferation . Am J Physiol Endocrinol Metabol . 2011 ; 301 ( 5 ): E864 - 72 .
31. Zhang B , Wang M , Gong A , Zhang X , Wu X , Zhu Y , Shi H , Wu L , Zhu W , Qian H , et al. HucMSC-exosome mediated-Wnt4 signaling is required for cutaneous wound healing . Stem Cells (Dayton, Ohio) . 2015 ; 33 ( 7 ): 2158 - 68 .
32. Song M , Lim J , Yu HY , Park J , Chun JY , Jeong J , Heo J , Kang H , Kim Y , Cho YM , et al. Mesenchymal stem cell therapy alleviates interstitial cystitis by activating Wnt signaling pathway . Stem Cells Dev . 2015 ; 24 ( 14 ): 1648 - 57 .
33. Leroux L , Descamps B , Tojais NF , Seguy B , Oses P , Moreau C , Daret D , Ivanovic Z , Boiron JM , Lamaziere JM , et al. Hypoxia preconditioned mesenchymal stem cells improve vascular and skeletal muscle fiber regeneration after ischemia through a Wnt4-dependent pathway . Mol Ther . 2010 ; 18 ( 8 ): 1545 - 52 .
34. Niehrs C. The complex world of WNT receptor signalling . Nat Rev Mol Cell Biol . 2012 ; 13 ( 12 ): 767 - 79 .
35. Ring L , Neth P , Weber C , Steffens S , Faussner A. beta-Catenin-dependent pathway activation by both promiscuous “canonical” WNT3a-, and specific “noncanonical” WNT4- and WNT5a-FZD receptor combinations with strong differences in LRP5 and LRP6 dependency . Cell Signal . 2014 ; 26 ( 2 ): 260 - 7 .
36. Breton-Romero R , Feng B , Holbrook M , Farb MG , Fetterman JL , Linder EA , Berk BD , Masaki N , Weisbrod RM , Inagaki E , et al. Endothelial dysfunction in human diabetes is mediated by Wnt5a-JNK signaling . Arterioscler Thromb Vasc Biol . 2016 ; 36 ( 3 ): 561 - 9 .
37. Fuster JJ , Zuriaga MA , Ngo DT , Farb MG , Aprahamian T , Yamaguchi TP , Gokce N , Walsh K. Noncanonical Wnt signaling promotes obesity-induced adipose tissue inflammation and metabolic dysfunction independent of adipose tissue expansion . Diabetes . 2015 ; 64 ( 4 ): 1235 - 48 .
38. Kim J , Kim J , Kim DW , Ha Y , Ihm MH , Kim H , Song K , Lee I. Wnt5a induces endothelial inflammation via beta-catenin-independent signaling . J Immunol (Baltimore , Md: 1950 ). 2010 ; 185 ( 2 ): 1274 - 82 .
39. Mounayar M , Kefaloyianni E , Smith B , Solhjou Z , Maarouf OH , Azzi J , Chabtini L , Fiorina P , Kraus M , Briddell R , et al. PI3kalpha and STAT1 interplay regulates human mesenchymal stem cell immune polarization. Stem Cells (Dayton , Ohio). 2015 ; 33 ( 6 ): 1892 - 901 .
40. Wang Y , Chen X , Cao W , Shi Y. Plasticity of mesenchymal stem cells in immunomodulation: pathological and therapeutic implications . Nat Immunol . 2014 ; 15 ( 11 ): 1009 - 16 .