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Erythropoietin Stimulates Endothelial Progenitor Cells to Induce Endothelialization in an Aneurysm Neck After Coil Embolization by Modulating Vascular Endothelial Growth Factor

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Significance: Erythropoietin (EPO) is involved in erythropoiesis and related conditions and is reported to enhance stem-cell mobilization from bone marrow while elevating stem-cell viability and function. The researchers report that EPO also stimulates endothelial progenitor cells to induce the endothelialization of a coiled embolic aneurysm neck via vascular endothelial growth factor modulation. Endothelialization induction provides an additional therapeutic opportunity during vascular inner layer repair and remodeling. The results provide important information on the unique role EPO plays during vascular repair and remodeling.
Experimental design, histological assessment, and SEM observation. (A): Rats with aneurysms treated with coil therapy and endpoint examination of FCM, SEM, H&E, and IHC, after EPO treatment. (B): Photomicrographs demonstrate the H&E-stained aneurysm neck in EPOtreated rats (a) and untreated rats with AN and (c) after 30 days of EPO administration. The integration of the AN neck can be observed in the magnified images (b, d). Scale bar = 50 mm. The bar graph shows the aneurysm repair score in the AN rat and the AN-EPO-treated rats (e). Data are means 6 standard errors of the mean; n = 17 per group; p, p , .05, EPO-AN rats vs. untreated rats. (C): Photomicrographs show the endothelialization in the AN neck under SEM. Consistent simple squamous epithelial cells at the bottom of the aneurysm neck were detected in the AN-EPO-treated rats (a) but not in AN rats (c). Scale bar = 200 mm. Magnified details shown in (b) and (d). Scale bar = 50 mm. (D): Photomicrographs show the vWF + and KDR + cells covering the bottom of the AN neck. The cells are stained with DAPI (blue), vWF (green in b, c, e, f), and KDR (h, i, k, l). The bar graph semiquantifies the vWF + and KDR + cell count per HPF in AN rats and AN-EPO rats (m, n). Data are means 6 standard errors of of the mean; n = 7 per group; p, p , .05, EPO-AN rats vs. untreated rats. Abbreviations: AA, abdominal aorta; AN, aneurysm; DAPI, 49,6-diamidino-2-phenylindole; EPO, erythropoietin; FCM, flow cytometry; H&E, hematoxylin and eosin; HPF, high power field; IF, immunofluorescence; IV, iliolumbar vein; RV, renal vein; SEM, scanning electron microscopy; vWF, von Willebrand factor.
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Endothelial progenitor cell (EPC) identification and erythropoietin (EPO)-induced cell death and viability changes. (A): Photomicrographs show the cultured EPCs that were used for EPC identification. Representative photomicrograph shows adherent primary cells after 10 days of culture (a, b magnified from a). Scale bar = 80 mm in (a), 40 mm in (b). Fluorescence photomicrographs showing Dil-ac-LDL (c), FITC-UEA-I (d), and a merged image (e). Scale bar = 40 mm. (B): Detection of CD34/KDR double-labeled cells (a), KDR cells (b), and CD34 cells (c) after 10 days of cell culture to identify EPCs. The bar graph shows a quantitative analysis of KDR + , CD34 + , and KDR + /CD34 + cells. (d) Data are the means 6 standard errors of the mean; n = 7 per group. (C): Representative photographs showing viable cells labeled by calcein acetoxymethyl and dead cells labeled by propidium iodide in cultured EPCs treated with 0.015 mg/l (b) and 0.15 mg/l (c), 1.5 mg/l (d), 15 mg/l (e), and 150 mg/l (f), compared with the control group (a). Scale bar = 200 mm. (D): The bar graph shows the changes in EPC viability changes after 1, 3, 7, and 10 days of EPO treatment. Data are means 6 standard errors of the mean; n = 5 per group; p, p , .05, EPO treatment group vs. control group. (E): Photomicrograph showing the migration of Hoechst-labeled EPCs (a-f) after EPO treatment cells (b-f) and under the control conditions (a). Scale bar = 200 mm. The bar graph shows EPC migration (g) after EPO treatment. Data are the means 6 standard errors of the mean; n = 5 per group; p, p , .05, EPO treatment group vs. control group. Abbreviation: UEA, Ulex europaeus.
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Tissue-Specific Progenitor and Stem Cells
Erythropoietin Stimulates Endothelial Progenitor
Cells to Induce Endothelialization in an Aneurysm
Neck After Coil Embolization by Modulating Vascular
Endothelial Growth Factor
PEIXI LIU,
a,
*
YINGJIE ZHOU,
b,
*
QINGZHU AN,
a
YAYING SONG,
c
XICHEN,
a
GUO-YUAN YANG,
c,d
WEI ZHU
a
Key Words. Cerebral aneurysm xEndothelialization xEndothelial progenitor cells xErythropoietin
ABSTRACT
This study explored a new approach to enhance aneurysm (AN) neck endothelialization via erythro-
poietin (EPO)-induced endothelial progenitor cell (EPC) stimulation. Results suggest that EPO en-
hanced the endothelialization of a coiled embolization AN neck by stimulating EPCs via vascular
endothelial growth factor modulation. Thus, the promotion of endothelialization with EPO provides
an additional therapeutic option for preventing the recurrence of ANs. Endovascular coil embolization
is an attractive therapy for cerebral ANs, but recurrence is a main problem affecting long-term out-
comes. In this study, we explored a new approach to enhance AN neck endothelialization via EPO-
induced EPC stimulation. Ninety adult male Sprague-Dawley rats were selected for an in vivo assay,
and 60 of them underwent microsurgery to create a coiled embolization AN model. The animals were
treated with EPO, and endothelial repair was assessed via flow cytometry, immunofluorescence, elec-
tronic microscopy, cytokine detection, and routine blood work. EPO improved the viability, migration,
cytokine modulation, and gene expression of bone marrow-derived EPCs and the results showed that
EPO increased the number of circulating EPCs and improved endothelialization compared with untreated
rats (p<.05). EPO had no significant effect on the routine blood work parameters. In addition, the im-
munofluorescence analysis showed that the number of KDR
+
cells in the AN neck was elevated in the
EPO-treated group (p<.05). Furtherstudy demonstrated that EPOpromoted EPC viability and migration
in vitro. The effects of EPO may be attributed to the modulation of vascular endothelial growth factor
(VEGF). In particular, EPO enhanced the endothelialization of a coiled embolization AN neck by stimu-
lating EPCs via VEGF modulation. Thus, the promotion of endothelialization with EPO provides an
additional therapeutic option for preventing the recurrence of ANs. STEM CELLS TRANSLATIONAL
MEDICINE 2016;5:18
SIGNIFICANCE
Erythropoietin (EPO) is involved in erythropoiesis and related conditions and is reported to enhance
stem-cell mobilization from bone marrow while elevating stem-cell viability and function. The re-
searchers report that EPO also stimulates endothelial progenitor cells to induce the endotheliali-
zation of a coiled embolic aneurysm neck via vascular endothelial growth factor modulation.
Endothelialization induction provides an additional therapeutic opportunity during vascular inner
layer repair and remodeling. The results provide important information on the unique role EPO plays
during vascular repair and remodeling.
INTRODUCTION
Compared with surgical management, endovas-
cular coil embolization is an attractive approach
for the treatment of unruptured, saccular cere-
bral aneurysms (ANs) because it is minimally in-
vasive and efficient [1]. However, endovascular
coil embolization has a high rate of recurrence
(6.1%33.6%) and rebleeding (0.11%1.6%) [26].
Because the lack of endothelialization plays a
crucial role in AN recurrence, the promotion of
endothelialization in the AN neck may help pre-
vent both recurrence and rupture.
Endothelial progenitor cells (EPCs) were first
identified from human peripheral blood and
are derived from the bone marrow. Circulating
EPCs promote endothelialization after coiled em-
bolization treatments, and EPC therapy has been
tested in many vascular diseases [7, 8]. Studies
have shown a correlation between a lack of EPCs
and the incidence of cerebral ANs and have also
demonstrated that bone marrow-derived EPCs
Departments of
a
Neurosurgery and
b
Hand
Surgery, Huashan Hospital of
Fudan University, Shanghai,
Peoples Republic of China;
c
Department of Neurology,
Ruijin Hospital, Shanghai Jiao
Tong University School of
Medicine, Shanghai, Peoples
Republic of China;
d
Neuroscience and
Neuroengineering Research
Center, Med-X Research
Institute and School of
Biomedical Engineering,
Shanghai Jiao Tong
University, Shanghai,
Peoples Republic of China
*
Contributed equally.
Correspondence: Wei Zhu, M.D.,
Ph.D., Huashan Hospital of Fudan
University, Department of
Neurosurgery, Wulumuqi Zhong
Road, Shanghai 200040, Peoples
Republic of China. Telephone: 86-
21-52887034; E-Mail: drzhuwei@
fudan.edu.cn; or Guo-Yuan Yang,
M.D., Ph.D., Neuroscience and
Neuroengineering Research
Center, Med-X Research Institute
and School of Biomedical
Engineering, Shanghai Jiao Tong
University, Shanghai 200030,
Peoples Republic of China,
Telephone: 86-21-62933186;
E-Mail: gyyang0626@gmail.com
Received September 27, 2015;
accepted for publication April 8,
2016.
©AlphaMed Press
1066-5099/2016/$20.00/0
http://dx.doi.org/
10.5966/sctm.2015-0264
STEM CELLS TRANSLATIONAL MEDICINE 2016;5:18 www.StemCellsTM.com ©AlphaMed Press 2016
T
ISSUE
-S
PECIFIC
P
ROGENITOR AND
S
TEM
C
ELLS
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are involved in the process of AN repair [9]. However, the contri-
bution of increased circulating EPCs after coiled embolization con-
tributing to AN neck endothelialization remains unverified.
Erythropoietin (EPO) is known for its function in erythropoiesis
and has been reported to enhance the mobilization of stem cells
from bone marrow and strengthen their viability and function.
EPO has shown therapeutic effects in the treatment of myocardial
infarcts, cerebral ANs, brain ischemia, and traumatic injuries [1012]
and may also promote angiogenesis to improve microcirculation
and neovascularization and protect neural tissue. EPO exhibits a
strong capacity to promote EPC differentiation and maturation to-
ward endothelial cell lineages. Increasingly, studies have strongly
indicated that EPO can reduce the formation and progression of
cerebral ANs by promoting EPC mobilization and targeting them
to sites of vascular injury [1315]. These findings support the hypoth-
esis that EPO may be used to protect against the recurrence of
cerebral AN by stimulating EPCs and promoting endothelialization.
We showed that EPCs enhanced AN neck endothelialization after
coil therapy and that the administration of EPO increased the num-
ber and function of EPCs. Because of the potency of EPO in promoting
EPCs, we explored the possibility of using EPO to promote AN neck
EPC-induced endothelialization in a coiled embolization AN model.
MATERIALS AND METHODS
All animal procedures were carried out according to a protocol that
was approved by the Institutional Animal Care and Use Committee
(IACUC), and the experimental protocol was reviewed and approved
by the Ethics Committee of Huashan Hospital affiliated with Fudan Uni-
versity in Shanghai, Peoples Republic of China. Adult Sprague-Dawley
rats (150200 g) were used in the experiments (Slac Laboratory Ani-
mal, Shanghai, Peoples Republic of China, http://english.sibs.cas.cn/
rs/fs/shanghailaboratoryanimalcentercas). The coiled AN model was
prepared as previously reported [16]. The AN-EPO group was admin-
istered 1.5 mg/kg Recombinant Rat EPO (R&D Systems, Minneapolis,
MN, https://www.rndsystems.com) injected subcutaneously, and the
AN group was given an equal amount of saline subcutaneously. The
mock surgery (MS) group was given inhalation anesthesia and was
treated with saline similarly to the AN group but without surgery.
On days 10, 20, and 30, peripheral blood was collected to examine cir-
culating EPCs via flow cytometry and changes in serum concentrations
of vascular endothelial growth factor (VEGF), tumor necrosis factor
(TNF)-a, and interleukin (IL)-6 via the MILLIPLEX MAP (EMD Millipore,
Billerica, MA, http://www.emdmillipore.com). On day 30, the AN tis-
sue was obtained for scanning electron microscopy, hematoxylin and
eosin (H&E) staining, and immunofluorescence. EPCs were isolated
from a healthy rat femur bone and cultured in EGM-2 medium (Lonza,
Anaheim, CA, http://www.lonza.com). EPCs were identified via Dil-ac-
LDL (Invitrogen, Carlsbad, CA, https://www.thermofisher.com)/FITC-
UEA-I (Sigma, San Louis, MO, http://www.sigmaaldrich.com) double
staining and vascular endothelial growthfactorreceptor2(VEGFR2,also
known as KDR; Abcam, Cambridge, MA, http://www.abcam.com/)/CD34
(R&D Systems, Minneapolis, MN, https://www.rndsystems.com) flow
cytometry analyses. The viability of EPO-treated cells was tested via a
Cell Counting Kit-8 (Dojindo Laboratories, Kumamoto, Japan, http://
www.dojindo.com), and the live and dead cells were distinguished us-
ing through calcein acetoxymethyl (AM) (AAT Bioquest, Sunnyvale,
CA, http://www.aatbio.com/) and propidium iodide (PI) (BD Pharmin-
gen, San Diego, CA, http://www.bdbiosciences.com) staining. The se-
creted VEGF, TNF-a, and IL-6 from cultured cells were analyzed by a
MILLIPLEX MAP. Gene expression was evaluated by quantitative
polymerase chain reaction (qPCR). The primer sequences are shown
in supplemental online Table 1.
Detailed methods are described in the online supplemental
data.
Statistical Analysis
The statistical analysis was performed using IBM SPSS Statistics
(Armonk, NY, http://www.ibm.com), and graphs were gener-
ated by GraphPad Prism (GraphPad, La Jolla, CA, http://www.
graphpad.com/company). Two-way ANOVA tests were used to
analyze the percent of circulating EPCs identified by flow cytom-
etry. Independent sample ttests were used to determine the an-
eurysm repair score, von Willebrand factor (vWF)
+
cell count,
KDR
+
cell count, and circulating cytokines [17]. One-way ANOVA
tests were used to analyze the peripheral blood, the optical den-
sity values obtained from the cell viability test, the migration cell
count, the levels of cytokines secreted from the cultured cells, and
the gene expression levels obtained by qPCR. Differences with
p,.05 were considered significant.
RESULTS
Experimental Design, Histological Assessment, and
Scanning Electron Microscopy Observations
After the coil embolism aneurysm model was initiated, 90 rats
survived until sacrifice. All coil embolism aneurysm specimens
were acquired for subsequent use.
H&E staining under low magnification demonstrated that a
more integrated aneurysm neck was formed, and more spindle-
like slender cells were observed in the aneurysm necks of the
AN-EPO-treated rats. In contrast, in the AN rats, fewer vascular en-
dothelial cells and only sparse fibrouscells were observed. The an-
eurysm repair score was significantly higher in the AN-EPO-treated
rats compared with the AN rats, p,.05 (Fig. 1B).
Scanning electron microscopy (SEM) examination revealed
the level of endothelialization in the aneurysm neck and found
better endothelial coverage in the AN-EPO-treated rats compared
with the AN rats. This layer primarily consisted of simple squa-
mous epithelial cells at the bottom of the aneurysm neck. Overall,
rats treated without EPO demonstrated a similar sealing effect
compared with the EPO-treated rats. However, endothelial cells
were rarely observed in the AN rats; instead, they displayed long,
flat, and fusiform cell morphologies (Fig. 1C).
Under confocal microscopy, consecutive and compact vWF
+
cells were found in the surface of the AN neck in the AN-EPO-
treated rats. It was noted that the vWF layer in the AN rats was
not continuous (Fig. 1D). There were significantly more vWF
+
and KDR
+
cells in the inner surface of the AN neck in the AN-
EPO-treated rats than in the AN rats, p,.05 (Fig. 1D).
Circulating EPC Detection and Peripheral Blood Changes
On day 10 after surgery, the circulating EPC count was signifi-
cantly elevated in the AN-EPO-treated rats compared with the
MS and AN rats (p,.05). On day 20, the circulating EPC count
significantly increased in the AN and AN-EPO groups compared
with the MS group. There was no indicated superiority in the
EPO treatment group. On day 30, the AN and AN-EPO-treated rats
continued to show to increase EPC counts, and this increase was
more obvious in the EPO-treated group (Fig. 2A).
2EPO Stimulates EPCs in an Aneurysm Neck
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We performed a peripheral blood examination to exclude the
possible side effect of EPO elevating the red blood cell (RBC)
count. We found no significant differences between the AN-EPO
group and the ANgroup for the RBC count, hemoglobin, red blood
cell specific volume, mean corpuscular volume, mean corpuscular
hemoglobin, and the mean corpuscular hemoglobin concentration
on days 10, 20, and 30 (Fig. 2B).
EPC Identification and Changes in EPO-Induced Cell
Death, Viability, and Migration
We isolated EPCs from the femur marrow and found that many
cells had a round, cobblestone-like morphology in the primary ad-
herent cell culture. Furthermore, most of these primary cells were
able to uptake both Dil-ac-LDL and FITC-UEA-I (Fig. 3A). Through
flow cytometry, we demonstrated that 62.96 64.48% of the cells
were KDR
+
,51.5363.65% of th e cells were CD34
+
,and33.9161.77%
of the cells were KDR/CD34
+
. The flow cytometry analyses indi-
cated that these KDR
+
/CD34
+
cells were EPCs (Fig. 3B).
PI and calcein AM-labeled cells were observed by confocal mi-
croscopy. No increase in the number of dead cells was observed in
the EPO-treated rats after 7 days of EPO treatment compared
with the control rats (Fig. 3C).
We recorded the absorbance of EPCs in each well after their
reaction with the reagents in a Cell Counting Kit-8. A significant
increase in absorbance was observed on days 7 and 10 using high
concentrations of EPO (150 mg/l and 15 mg/l), and an EPO concen-
tration of 1.5 mg/l also presented a significant increase in absor-
bance on day 10. These findings indicate that EPCs presented
improved cell viability when cultured with high concentrations
of EPO for prolonged durations (Fig. 3D).
We performed a scratch assay to assess the degree of EPC migra-
tion and found that treatments with 150 mg/l, 15 mg/l, and 1.5 mg/l
EPO for 10 days significantly increased EPC migration, p,.05 (Fig. 3E).
Figure 1. Experimental design, histological assessment, and SEM observation. (A): Rats with aneurysms treated with coil therapy and endpoint
examination of FCM, SEM, H&E, and IHC, after EPO treatment. (B): Photomicrogr aphs demonstrate the H&E-stained aneurysm neck in EPO-
treated rat s (a) and untreated rats w ith AN and (c) after 30 days o f EPO administration. The in tegration of the AN neck ca n be observed in the
magnified images (b, d). Scale bar = 50 mm. The bar grap h shows the aneurysm re pair score in the AN rat and the A N-EPO-treated ra ts (e). Data
are means 6standard errors of the mean; n= 17 per group; p,p,.05, EPO-AN rats vs. untreated rats. (C): Photomicrographs show the
endotheli alization in the AN nec k under SEM. Consisten t simple squamous epi thelial cells at the bot tom of the aneurysm nec k were detected
in the AN-EPO-treated rats (a) but not in AN rats (c). Scale bar = 200 mm. Magnified details shown in (b) and (d). Scale bar = 50 mm. (D):
Photomicrographs show the vWF
+
and KDR
+
cells covering the bottom of the AN neck. The cells are stained with DAPI (blue), vWF (green
in b, c, e, f), and KDR (h, i, k, l). Thebar graph semiquantifies the vWF
+
and KDR
+
cell count p er HPF in AN rats and AN-EP O rats (m, n). Data are
means 6standard errors of of the mean; n=7pergroup;p,p,.05,EPO-AN rats vs. untreated rats. Abbreviations: AA, abdo minal aorta; AN,
aneurysm; DAPI, 49,6-diamidino-2-phenylindole; EPO, erythropoietin; FCM, flow cytometry; H&E, hematoxylin and eosin; HPF, high power
field; IF, immunofluorescence; IV, iliolumbar vein; RV, renal vein; SEM, scanning electron microscopy; vWF, von Willebrand factor.
Liu, Zhou, An et al. 3
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Cerebrovascular Cytokine Levels and Gene Expression
After EPO Treatment
We tested cytokines and chemokines in the peripheral blood in
both the AN and AN-EPO groups, including VEGF, TNF-a, and
IL-6. The peripheral blood VEGF concentration in the AN-EPO
group was elevated on days 20 and 30 compared with the AN
group. There was no significant differences in the TNF-aand
IL-6 levels between the AN and AN-EPO-treated rats (Fig. 4A).
However, significant changes in cytokines and chemokines
levels were observed in vitro. On day 1, the levels of VEGF from
EPO-treated cells were significantly increased, and the IL-6 level
was decreased (Fig. 4B). On day 3, the VEGF levels of the EPCs
treated with 150 mg/l, 15 mg/l, 0.15 mg/l, and 0.015 mg/l EPO were
significantly higher than those observed in the control group. The
reducing effect of EPO on the IL-6 levels was not obvious, and only
150 mg/l EPO induced inhibition. There were no significant changes
in the TNF-alevels on either day 1 or day 3 (Fig. 4B).
qPCR analysis was used to determine the VEGF, TNF-a,and
IL-6 gene expression levels. After 1 and 3 days of 150 mg/l EPO
treatment, the expression of VEGF showed evident elevation,
Figure 2. Circulating EPC detection, cytokine and chemokine evaluation, and peripheral blood changes. (A): Circulating EPCs were counted on
days 10, 20, and 30 in MS rats (a, d, g), untreated AN rats (b, e, h), and AN-EPO-treated rats (c, f, i). The bar graph displays quantified CD34
+
/KDR
+
cells in MS rats, AN rats, and AN-EPO rats (j). Data are means 6standard errors of the mean; n= 10 per group; p,p,.05, EPO-AN group vs.
untreated group and MS group. (B): Rat peripheral blood tests from MS, AN, and AN-EPO rats. The RBC (a), HGB (b), HCT (c), MCV (d), MCH (e),
and MCHC (f) are shown in the bar graph. Data are means 6standard errors of the mean; n= 10 per group. Abbreviations: AN, aneurysm; EPC,
endothelial progenitor cell; EPO, erythropoietin; HCT, red blood cell specific volume; HGB, hemoglobin; MCH, mean corpuscular hemoglobin;
MCHC, mean corpuscular hemoglobin concentration; MCV, mean corpuscular volume; MS, mock surgery; Q, quadrant; RBC, red blood cell.
4EPO Stimulates EPCs in an Aneurysm Neck
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and IL-6 showed gradually decreasing trend. No significant dif-
ferences were observed for the TNF-aand IL-6 gene expression
levels (Fig. 4C).
DISCUSSION
In our study, EPO was administered after the induction of a coiled
embolization AN to prevent recurrence. We successfully established
a coiled AN rat model via vasotransplantation. Half of the AN rats
were treated with EPO. This EPO-treated group showed a significant
increase in the number of circulating EPCs in the rats with aneu-
rysms after coiling, and the EPCs also participated in the aneu-
rysm neck endothelialization. EPO promoted the AN neck
integration and endothelialization, as shown by SEM. We also
found that in vitro bone marrow-derived EPCs could be en-
hanced after 7 days of EPO treatment. Our study showed that
EPO increased VEGF levels in vivo and in vitro. The safety of
short-term EPO treatment was indicated by showing fewer side
effects on the AN rat peripheral blood work, such as increased
RBC counts or EPC deaths in vitro.
Figure 3. Endothelial progenitor cell (EPC) identification and erythropoietin (EPO)-induced cell death and viability changes. (A): Photomicro-
graphs show the cultured EPCs that were used for EPC identification. Representative photomicrograph shows adherent primary cells after 10
days of culture (a, b magnified from a). Scale bar = 80 mm in (a), 40 mm in (b). Fluorescence photomicrographs showing Dil-ac-LDL (c), FITC-UEA-I
(d), and a merged image (e). Scale bar = 40 mm. (B): Detection of CD34/KDR double-labeled cells (a), KDR cells (b), and CD34 cells (c) after
10 days of cell cultureto identify EPCs. Thebar graph shows a quantitative analysisof KDR
+
,CD34
+
, and KDR
+
/CD34
+
cells. (d) Dataare the means 6
standard errors of the mean; n= 7 per group. (C): Representative photo graphs showing viable cells labeled by calcein acetoxymethyl and dead
cells labeled by propidium iodide in cultured EPCs treated with 0.015 mg/l (b) and 0.15 mg/l (c), 1.5 mg/l (d), 15 mg/l (e), and 150 mg/l (f),
compared with the control group (a). Scale bar = 200 mm. (D): The bar graph shows the changes in EPC viability changes after 1, 3, 7, and 10
days of EPO tre atment. Data are mean s 6standard errors of t he mean; n= 5 per group; p,p,.05, EPO treatment group vs. control group. (E):
Photomicrograph showing the migration of Hoechst-labeled EPCs (af) after EPO treatment cells (bf) and under the control conditions (a).
Scalebar=200mm. The bar graph shows EPC migration (g) after EPO treatment. Data are the means 6standard errors of the mean; n= 5 per
group; p,p,.05, EPO treatment group vs. control group. Abbreviation: UEA, Ulex europaeus.
Liu, Zhou, An et al. 5
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Figure 4. Cerebrovascular cytokines level and gene expression after EPO treatment in cultured endothelial progenitor cells (EPCs). (A): Bar
graph demonstrates the peripheral serum levels of VEGF (a), TNF-a(b), and IL-6 (c). Data are means 6standard errors of the mean; n=8
per group; p,p,.05, EPO-AN rats vs. untreated rats. (B): VEGF (a, d), TNF-a(b, e), and IL-6 (c, f) from cultured EPCs were measured on days
1 and 3. Data are means 6standard errors of the mean; n= 3 per group; p,p,.05 compared with untreated group. (C): Bar graph demonstrates
the levels of VEGF (a), TNF-a(b), and IL-6 (c) expression of EPCs after 150 mg/l EPO treatments for 1 day and 3 days. Data are means 6standard
(Figure legend continues on next page.)
6EPO Stimulates EPCs in an Aneurysm Neck
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Endothelialization in the AN neck has been observed in clinical
studies [18, 19] and animal models [20]. Endothelial dysfunctions
are regarded as the main contributor to the recurrence of postem-
bolization cerebral AN. The promotion of endothelialization is key to
preventing AN recurrence. Because EPCs were initially identified
from human peripheral blood and found to express CD34 and KDR
[21], EPC therapy was used to facilitate vascular repair and homeo-
stasis [21, 22]. The autologous transfusion of bone marrow-derived
EPCs could be used for stroke treatment [23]. There is a growing in-
terest in using circulating EPCs in clinical trials for ischemia, stroke,
and vascular injury [2427]. Our previous study[16] also showed that
bone marrow-derived EPCs play a crucial role in the closure and re-
construction of the aneurysm neck orifice after aneurysm coiling.
Medications, including statins, angiotensin converting en-
zyme inhibitors, and cancer drugs, have been studied to deter-
mine their effects on EPC mobilization. EPO has been shown to
reduce the formation and progression of cerebral aneurysms in
rats [28]. In this study, we found that the number of circulating
EPCs was significantly increased at 10 days after EPO treatment
in coiled AN rats compared with nontreated AN rats, which could
be caused by mobilization promoted by EPO during early stages.
On day 20, both the AN and AN-EPO-treated rats induced EPC mo-
bilization and homing after vascular injury. During this stage, the
effect of EPO did not appear to be significant. Late stages of endo-
thelialization were evident on day 30. In these stages, the number
of AN neck EPCs decreased, and the circulating EPC levels recov-
ered to a relatively high level. During this period, the EPO-treated
rats maintained an obviously higher number of circulating EPCs.
Previous studies have shown strong evidence indicating that EPO
can mobilize EPCs from bone marrow to increase the amount of
circulating EPCs [28,29]. During the AN neck endothelialization, the
circulating EPCs of the AN-EPO group were significantly increased
significantly on day 10. With the completion of endothelialization,
the circulating EPCs showeda decrease trend on days 20 and 30. In
contrast, the circulating EPCs of the rats belonging to the AN group
showed a significant increase on day 20 comparedwith the EPCs of
the MS groupand showed a decreasing trend in day30. These find-
ings indicate that EPO maylead to earlier mobilization. However, it
is possible that the time interval between EPO administration and
blood collection may partially affect the circulating EPCs.
EPO is a hormone secretedby the kidney in response to hypoxia
and plays a cardinal role in regulating plasma hemoglobin concen-
trations.Accordingto routine bloodtests, the shortterm use of EPO
did not show any side effects in the AN-EPOrats. In in vitro EPC cul-
tures, EPO didnot show significant cytotoxicity.In a previous study,
EPO showed vascular protection and endothelium-promoting
properties. These effects were primarily mediated by inhibiting ap-
optosis, mobilizing endothelial progenitor cells, inhibiting the mi-
gration of inflammatory cells, and promoting angiogenesis [12].
Previous studies on EPO-mediated endothelialization and its mo-
lecular pathways have focused on antiapoptotic and survival sig-
nals, including the phosphatidylinositol 3-kinase pathway and
the endothelial nitricoxide synthase pathway [3032].In our study,
we assessed the levelsof VEGF, TNF-aandIL-6. We foundthat VEGF
increasedafter EPO treatment in vivoand in vitro. The modulation
of VEGF by EPO isthought to be one of the most important factors
in promoting AN neck endothelialization after coil embolization
treatment. EPO modulation plays key roles via VEGF and VEGF re-
ceptors in many vascular diseases [3235].
In EPC cultures treated with EPO for 1 and 3 days, the levels of
IL-6 showeda decreasingtrend. This may be attributed to the anti-
inflammatory effect of EPO. However, the in vivo IL-6 levels of AN
rat sera and the in vitro gene expression profile of IL-6 did not cor-
relate with this observation. This phenomenon may be induced by
complex cytokine regulation and would require further study. Fur-
thermore, no strong evidence or discernible trend was observed
linking EPO and TNF-ain coiled AN rat sera or cultured EPCs.
CONCLUSION
In its recombinant form, EPO has been tested in clinical trials and
proven to be beneficial in cerebral vascular diseases by providing vas-
cular protection, inhibiting inflammation, and promoting endotheli-
alization [3638]. This study represents the first use of recombinant
rat EPO to promote coiled AN neck endothelialization. We showed
that EPO enhanced the endothelialization of coiled AN neck via VEGF
modulation. EPO or its analogs may provide new therapeutic alter-
natives in preventing recurrence in coiled cerebral AN. However,
there remain limitations. The peripheral blood cytokine levels may
fluctuate in part owing to the deferent interval between administra-
tion and blood collection. No extensive dose response studies were
performed in animal models. The exact and detailed mechanism
through which EPO affects endothelialization remains unclear. Other
important factors in addition to VEGF may be involved, such as hyp-
oxia inducible factor 1 and stromal-derived factor 1, which are also
known to mobilize EPCs under conditions of vascular disease and in-
jury [3941]. Further studies are required to explore the mechanism
responsible for the EPO-based endothelialization of aneurysm necks.
ACKNOWLEDGMENTS
This project was sponsored by Natural Science Foundation of
China (NSFC) Project No. 30973105 (W.Z.) and NSFC Project No.
81471178 (G.-Y.Y.), the Program for New Century Excellent Tal-
ents in University Project No. XYQ2011019 (W.Z.), and the Shang-
hai Rising Star Program Project No. 13QH1400900 (W.Z.).
AUTHOR CONTRIBUTIONS
P.L.: conception and design of the study, provision of study materials
and animal models, collection and/or assembly of data, data analysis
and interpretation, manuscript writing, final approval of manuscript;
Y.Z.: animal model creation, blood sample analysis, histological exam-
ination, discussion of manuscript; Q.A.: animal model analysis, histo-
logical examination; Y.S.: data analysis and interpretation, manuscript
writing; X.C.: database input, data interpretation; G.-Y.Y.: provision of
study material or patients, revision and final approval of manuscript;
W.Z.: conception and design, revision and final approval of manuscript.
DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
The authors indicated no potential conflicts of interest.
(Figure legend continued from previous page.)
errors of the mean; n= 3 per group; p,p,.05 compared with untreated group. Abbreviations: AN, aneurysm; EPO, erythropoietin; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; IL-6, interleukin 6; TNF-a, tumor necrosis factor a; VEGF, vascular endothelial growth factor.
Liu, Zhou, An et al. 7
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8EPO Stimulates EPCs in an Aneurysm Neck
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... 23 A similarly beneficial effect was observed in rats treated with erythropoietin, and this effect was potentially mediated by increased EPC expression of vascular endothelial growth factor. 24 Finally, Yu and colleagues showed that injection of microRNA-31a-5p also increased circulating EPC levels and improved aneurysm neck endothelialization relative to control animals. These effects were found to potentially be mediated by the effect of Axin on the Wnt/β-catenin molecular pathway. ...
... 25 In these studies, the degree of aneurysm occlusion was reported as a score quantifying both the degree of dome recanalization and neck endothelialization, with higher scores indicating more complete occlusion. [23][24][25] Absolute complete occlusion rates between intervention and control groups were not provided, nor were the diameter and length of coil inserted into the aneurysms. Taken together, these preclinical studies suggest that the modulation of circulating levels of EPCs holds promise as a possible adjunct to endovascular coiling. ...
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Endothelial progenitor cell (EPC) recruitment and angiogenesis play crucial roles in aneurysm neck endothelialization, but the mechanisms of EPC recruitment and angiogenesis are still unclear. Recent studies have shown that long noncoding RNAs (lncRNAs) can regulate the function and differentiation of cells in various ways. LncRNA TUG1 is involved in liver cancer and glioma-mediated angiogenesis. The aim of this study was to investigate the role of lncRNA TUG1 in regulating EPC migration and differentiation. Overexpression and knockdown of lncRNA TUG1 with lentivirus, scratch assays, Transwell assays and tube formation assays using EPCs isolated from rat bone marrow showed that lncRNA TUG1 overexpression promoted EPC migration, invasion and differentiation. Moreover, ELISAs showed that lncRNA TUG1 overexpression increased VEGF expression. Bioinformatics prediction, luciferase assays, Western blots and RIP assays indicated that lncRNA TUG1 functions as a ceRNA (competing endogenous RNA) for miR-6321 and that miR-6321 inhibits EPC migration and differentiation through its target, ATF2. As a potential therapeutic target, lncRNA TUG1 may play a vital role in the pathogenesis of aneurysms.
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Intracranial aneurysm (IA) is a complex disease resulting in subarachnoid hemorrhage (SAH) due to a rupture. The average worldwide prevalence of this disease is about 2–5 %, with 50 % of them ending in death or neurological disorders of varying severity, with a high probability of recurrence of hemorrhage during the frst half of the year after rupture. Subarachnoid hemorrhage is annually registered in at least 18 thousand people in Russia. Associations of polymorphic variants rs594942 and rs11603042 of the VEGFB gene in intracranial aneurysm development in the Volga-Ural region of the Russian Federation with the presence of the symptom complex of undifferentiated connective tissue dysplasia (uDST) and arterial hypertension (AH) were investigated. The C * allele rs594942 and rs11603042 of the VEGFB gene is a marker of an increased risk of IA as a whole ( p = 0.025; χ2 = 5.052; OR = 1.32) in women as a whole ( p = 0.001; χ2 = 10.124; OR = 1.70) and in comorbid state with uDCT ( p = 0.002; χ2 = 9.501; OR = 2.34) and AG ( p = 0.006; χ2 = 7.385; OR = 2.109). We found that the genotype * C * C of locus rs594942 of the VEGFB gene is a marker of an increased risk of intracranial aneurysm in general ( p = 0.017; χ2 = 5.702; OR = 1.49) and among women in general ( p = 0.0005; χ2 = 12.078; OR = 2.25) and with the symptomatic complex uCTD ( p = 0.007; χ2 = 7.173; OR = 2.67) and AH ( p = 0.010; χ2 = 6.471; OR = 2.51). We have obtained new results on the role of polymorphic variants of the VEGFB gene in the formation of intracranial aneurysm, taking into account the presence of the symptom complex uDCT and AH among the residents of the Volga-Ural region of Russia. A burdened comorbid background and the presence of undifferentiated connective tissue dysplasia and arterial hypertension can contribute to an increased risk of intracranial aneurysm, as evidenced by the results of our study.
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Endothelial progenitor cells (EPCs) have the ability to form new blood vessels and protect ischemic tissues from damage. We previously reported that EPCs with low activity of aldehyde dehydrogenase (Alde-Low EPCs) possess the greater ability to treat ischemic tissues compared with Alde-High EPCs. The expression level of the hypoxia inducible factors, HIF-1α and HIF-2α, was found to be greater in Alde-Low EPCs than in Alde-High EPCs. However, the precise role of the HIF factors in the regulation of EPC activity remains obscure. Here, we demonstrate a critical role of HIF-2α and its target gene CXCR4 for controlling the migratory activity of EPC to ischemic tissue. We found that co-culture of Alde-High EPCs with microvesicles (MVs) derived from Alde-Low EPCs improved their ability to repair an ischemic skin flap, and the expression of CXCR4 and its ligand SDF1 was significantly increased following the co-culture. In Alde-Low EPCs, the expression of CXCR4 was suppressed by shRNA-mediated HIF-2α, but not HIF-1α downregulation. Chromatin immunoprecipitaion assays showed that HIF-2α, but not HIF-1α, binds to the promoter region of CXCR4 gene. The CXCR4 shRNA treatment in Alde-Low EPCs almost completely abrogated their migratory activity to ischemic tissues, whereas the reduction of vascular endothelial growth factor (VEGF) showed much less effect. The CXCR4 overexpression in Alde-High EPCs resulted in a partial, but significant improvement in their repairing ability in an ischemic skin flap. Collectively, these findings indicate that the CXCR4/SDF-1 axis, which is specifically regulated by HIF-2α, plays a crucial role in the regulation of EPC migration to ischemic tissues.
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Background: Cerebral arteriovenous malformation (AVM) involves the vasculogenesis of cerebral blood vessels and can cause severe intracranial hemorrhage. Stromal cell-derived factor-1 (SDF-1) and its receptor, CXCR4, are believed to exert multiple physiological functions including angiogenesis. Thus, we investigated the role of SDF-1/CXCR4 in the vasculogenesis of cerebral AVM. Methods: Brain AVM lesions from surgical resections were analyzed for the expression of SDF-1, CXCR4, VEGF-A, and HIF-1 by using immunohistochemical staining. Flow cytometry was used to quantify the level of circulating endothelial progenitor cells (EPCs). Further, in an animal study, chronic cerebral hypoperfusion model rats were analyzed for the expression of SDF-1 and HIF-1. CXCR4 antagonist, AMD3100, was also used to detect its effects on cerebral vasculogenesis and SDF-1 expression. Results: Large amounts of CXCR4-positive CD45(+) cells were found in brain AVM lesion blood vessel walls, which also have higher SDF-1 expression. Cerebral AVM patients also had higher level of EPCs and SDF-1. In chronic cerebral hypoperfusion rats, SDF-1, HIF-1, and CD45 expressions were elevated. The application of AMD3100 effectively suppressed angiogenesis and infiltration of CXCR4-positive CD45(+) cells in hypoperfusion rats compared to controls. Conclusion: The SDF-1/CXCR4 axis plays an important role in the vasculogenesis and migration of inflammatory cells in cerebral AVM lesions, possibly via the recruitment of bone marrow EPCs.
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Hyperbaric oxygen (HBO) therapy has been used as an adjunctive therapy for diabetic foot ulcers, although its mechanism of action is not completely understood. Recently, it has been shown that HBO mobilizes the endothelial progenitor cells (EPCs) from bone marrow that eventually will aggregate in the wound. However, the gathering of the EPCs in diabetic wounds is impaired due to the decreased levels of local stromal-derived factor-1α (SDF-1α). Therefore, we investigated the influence of HBO on HIF-1, which is a central regulator of SDF-1α and is down-regulated in diabetic wounds. The effects of HBO on HIF-1α function were studied in human dermal fibroblasts, SKRC7 cells and HIF-1α knock-out and wild-type mouse embryonic fibroblasts using appropriate techniques (Western blot, Quantitative-PCR and Luciferase hypoxia-responsive element (HRE) reporter assay). Cellular proliferation was assessed using H3-thymidine incorporation assay. The effect of HIF in combination with HBOT was tested by inoculating stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in db/db mice exposed to HBO.HBO activates HIF-1α at several levels by increasing both HIF-1α stability (by a non-canonical mechanism) and activity (as show both by induction of relevant target-genes and by a specific reporter assay). HIF-1α induction has important biological relevance because the induction of fibroblast proliferation in HBO disappears when HIF-1α is knocked down. Moreover, the local transfer of stable HIF-1α-expressing adenovirus (Adv-HIF) into experimental wounds in diabetic (db/db mice) has an additive effect on HBO-mediated improvements in wound healing.In conclusion HBO stabilizes and activates HIF-1, which contributes to increased cellular proliferation. In diabetic animals, the local transfer of active HIF further improves the effects of HBO on wound healing.
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Many studies have demonstrated beneficial effects of either erythropoietin (EPO) or endothelial progenitor cell (EPC) treatment in cerebral ischemia. To improve post-ischemic tissue repair, we investigated the effect of systemic administration of endothelial colony-forming cells (ECFCs), considered as relevant endothelial progenitors due to their specific vasculogenic activity, in the presence or absence of EPO, on functional recovery, apoptosis, angiogenesis, and neurogenesis in a transient focal cerebral ischemia model in the adult rat. Experimental study. The rats were divided into four groups 24 hours after ischemia,, namely control, ECFCs, EPO, and ECFCs+EPO, and received a single intravenous injection of ECFCs (5×10(6) cells) and/or intraperitoneal administration of EPO (2500 UI/kg per day for 3 days). Infarct volume, functional recovery, apoptosis, angiogenesis, and neurogenesis were assessed at different time points after ischemia. The combination of EPO and ECFCs was the only treatment that completely restored neurological function. The ECFCs+EPO treatment was also the most effective to decrease apoptosis and to increase angiogenesis and neurogenesis in the ischemic hemisphere compared to controls and to groups receiving ECFCs or EPO alone. These results suggest that EPO could act in a synergistic way with ECFCs to potentiate their therapeutic benefits.
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Erythropoietin (EPO) was hypothesized to mitigate reperfusion injury, in part via mobilization of endothelial progenitor cells (EPCs). The REVEAL trial found no reduction in infarct size with a single dose of EPO (60,000 U) in patients with ST-segment elevation myocardial infarction. In a substudy, we aimed to determine the feasibility of cryopreserving and centrally analyzing EPC levels to assess the relationship between EPC numbers, EPO administration, and infarct size. As a prespecified substudy, mononuclear cells were locally cryopreserved before as well as 24 and 48-72 h after primary percutaneous coronary intervention. EPC samples were collected in 163 of 222 enrolled patients. At least one sample was obtained from 125 patients, and all three time points were available in 83 patients. There were no significant differences in the absolute EPC numbers over time or between EPO- and placebo-treated patients; however, there was a trend toward a greater increase in EPC levels from 24 to 48-72 h postintervention in patients receiving ≥30,000 U of EPO (P = 0.099 for CD133(+) cells, 0.049 for CD34(+) cells, 0.099 for ALDH(br) cells). EPC numbers at baseline were inversely related to infarct size (P = 0.03 for CD133(+) cells, 0.006 for CD34(+) cells). Local whole cell cryopreservation and central EPC analysis in the context of a multicenter randomized trial is feasible but challenging. High-dose (≥30,000 U) EPO may mobilize EPCs at 48-72 h, and baseline EPC levels may be inversely associated with infarct size.