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Endothelial progenitor cell transplantation improves long-term stroke outcome in mice.

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ORIGINAL ARTICLE
Endothelial Progenitor Cell
Transplantation Improves Long-Term
Stroke Outcome in Mice
Yongfeng Fan, PhD,1 Fanxia Shen, MD,1,2 Tim Frenzel, PhD,1,3
Wei Zhu, MD,1 Jianqin Ye, MD,4 Jianrong Liu, MD,1,2
Yongmei Chen, PhD,1 Hua Su, MD,1 William L. Young, MD,1,5,6
and Guo-Yuan Yang, MD, PhD1,2,7
Objective: Endothelial progenitor cells (EPCs) play an important role in tissue repairing and regeneration in
ischemic organs, including the brain. However, the cause of EPC migration and the function of EPCs after ischemia
are unclear. In this study, we demonstrated the effects of EPCs on ischemic brain injury in a mouse model of
transient middle cerebral artery occlusion (tMCAO).
Methods: Circulating human EPCs were characterized with immunofluorescent staining and flow cytometry. EPCs
(1 ⫻ 106) were injected into nude mice after 1 hour of tMCAO. Histological analysis and behavioral tests were
performed from day 0 to 28 days after tMCAO.
Results: EPCs were detected in ischemic brain regions 24 hours after tMCAO. EPC transplantation significantly
reduced ischemic infarct volume at 3 days after tMCAO compared with control animals ( p ⬍ 0.05). CXCR4 was
expressed in the majority of EPCs, and stromal-derived factor-1 (SDF-1) induced EPC migration, which was blocked
by pretreated EPCs with AMD3100 in vitro. SDF-1 was upregulated in ischemic brain. Compared with control
animals, injecting AMD3100-pretreated EPCs resulted in a larger infarct volume 3 days after tMCAO, suggesting
that SDF-1–mediated signaling was involved in EPC-mediated neuroprotection. In addition, EPC transplantation
reduced mouse cortex atrophy 4 weeks after tMCAO and improved neurobehavioral outcomes ( p ⬍ 0.05). EPC
injection potently increased angiogenesis in the peri-infarction area ( p ⬍ 0.05).
Interpretation: We conclude that systemic delivery of EPCs protects the brain against ischemic injury, promotes
neurovascular repair, and improves long-term neurobehavioral outcomes. Our data suggest that SDF-1–mediated
signaling plays a critical role in EPC-mediated neuroprotection.
ANN NEUROL 2010;67:488 – 497
C
irculating endothelial progenitor cells (EPCs) are regarded as the cells expressing both human stem cell
markers CD34 or CD133, and endothelial cell markers
CD31, KDR, von Willebrand factor (vWF), vascular endothelial (VE)-cadherin, and Tie2, serving as a cell reservoir to maintain vascular homeostasis by monitoring and
repairing dysfunctional endothelium.1,2 Since their discovery in circulating blood,3 EPCs have been a hot topic
because of their potential in clinical applications. The ef-
fects of EPCs have broken the dogma that revascularization only occurs during embryonic development, and injection of EPCs into ischemic limbs has initiated de novo
vascularization.3 Long-term studies indicate that EPC
number/function in peripheral blood is an independent
risk marker for future cardiovascular events.4 Animal
models and human clinical studies indicate that transplantation of EPCs could improve functional recovery of heart
and limbs after ischemic injury.5–9
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.21919
Received Aug 22, 2009, and in revised form Oct 20, 2009. Accepted for publication Oct 30, 2009.
Address correspondence to Dr Yang, Neuroscience and Neuroengineering Center, Med-X Research Institute. Shanghai Jiao Tong University, 1954
Hua Shan Road, Shanghai 200030, China. E-mail: gyyang1@gmail.com
2
From the 1Center for Cerebrovascular Research, Department of Anesthesia and Perioperative Care, University of California, San Francisco, CA;
Department of Neurology, Ruijin Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China; 3Department of Anesthesiology
and Intensive Care, University Hospital, Münster, Germany; 4Department of Internal Medicine, 5Department of Neurological Surgery, and
6
Department of Neurology, University of California, San Francisco, CA; and 7Neuroscience and Neuroengineering Center, Med-X Research
Institute, Shanghai Jiao Tong University, Shanghai, China.
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© 2010 American Neurological Association
Fan et al: EPCs and Recovery
Ischemic stroke is a leading cause of mortality and
morbidity in the world. Currently, there are still no effective therapies available despite some promising advances
in basic stroke research.10,11 The advance of cell therapy
in cardiovascular diseases suggests that EPC transplantation is a promising option for the treatment of ischemic
brain injury. Patients with a higher number of circulating
EPCs after stroke have better outcomes than those with
fewer EPCs,12–14 suggesting that EPC may serve as a new
marker for stroke outcomes. EPCs also participate in revascularization after focal cerebral ischemia.15 EPCs isolated from bone marrow (BM) attenuate ischemic injury
in rats.16 However, it is unclear whether revascularization
mediated by EPCs improves long-term neurobehavioral
outcomes after ischemic stroke.
In the present studies, we administrated human
EPCs (hEPCs) intravenously to adult nude mice to explore whether hEPCs are able to home to ischemic regions and whether hEPC therapy results in improvement
of neurobehavioral outcomes. Furthermore, we investigated whether hEPC homing is mediated by stromalderived factor-1 (SDF-1)/CXCR4 signaling.
Materials and Methods
EPCs Isolation and Enrichment
Studies involving humans were institutional review boardapproved, and procedures for the use of laboratory animals were
approved by the Institutional Animal Care and Use Committee
(University of California, San Francisco). Isolation and characterization of EPCs were described in our previous study.17
Immunocytology and Immunohistochemistry
Mononuclear cells were plated on fibronectin-coated chamber
slides. After 5 days of culture, putative EPCs were extensively
washed with phosphate-buffered saline (PBS) and stained with
2.5␮g/ml of DiI-labeled acetylated low-density lipoprotein (Cell
Systems, Kirkland, WA) at 37°C for 30 minutes, followed by
fluorescein isothiocyanate (FITC)-Ulex europeus agglutinin
(UEA) staining (Sigma, St. Louis, MO). For characterization,
EPCs were incubated in primary antibodies: vWF (1:500;
Chemicon, Temecula, CA), VE-cadherin (1:500; Santa Cruz
Biotechnology, Santa Cruz, CA), KDR (1:200; Santa Cruz Biotechnology), and Tie2 (1:200; Santa Cruz Biotechnology). Second antibodies conjugated with fluorescence (Molecular Probes,
Eugene, OR) were applied. Isotype-specific antibodies (BD Biosciences, San Jose, CA) were used as negative controls. For brain
sections, the primary antibodies (SDF-1, 1:100, Upstate, Charlottesville, VA; CD31, 1:200, BD Bioscience; CD34, 1:200, BD
Pharmingen, Heidelberg, Germany) were applied overnight at
4°C. The secondary antibodies were Alexa Fluor 594, Alexa
Fluor 488, or Cy3. Peroxidase-conjugated streptavidin and substrate were used for detection. Negative controls were performed
by omitting the primary antibodies.
April, 2010
Flow Cytometry Analysis
EPC markers were analyzed using a flow cytometry (FACScalibur, BD Biosciences). EPCs were incubated with fluorescenceconjugated mouse antihuman monoclonal antibodies in the dark
for 20 minutes (CD34-PE, KDR-APC, VE-cadherin-FITC,
CD31-PE, CD11b-APC, CD14-PerCP, CXCR4-APC, BD Biosciences). The cells were then washed 3 times with fluorescenceactivated cell sorting (FACS) buffer, fixed with 2% paraformaldehyde and resuspended in FACS buffer. Human
immunoglobulin G (IgG) was used as negative controls. For
each sample, 5,000 events were analyzed.
Transient Middle Cerebral Artery
Occlusion Models
Adult nude CD-1 mice (Charles River, Wilmington, MA)
weighing 30 –35g were subjected to transient middle cerebral artery occlusion (tMCAO). Middle cerebral artery was occluded
using 6-0 nylon suture (Ethicon, Somerville, NJ) for 1 hour.
Reperfusion was performed by removing the suture.
hEPCs (1 ⫻ 106) were systemically injected into these
nude mice through the left jugular vein 1 hour after tMCAO.
Control animals received the same amount of human brain endothelial cells (HBECs) or saline. DiI-labeled hEPCs and
HBECs were used for cell tracking in ischemic brain. All 3
groups were sacrificed following 3 days or 4 weeks of reperfusion.
Behavioral Tests
Animals underwent 3 behavioral tests before tMCAO, and 1, 7,
14, and 28 days after tMCAO. Mice were trained for 3 days
before tMCAO with 3 consecutive trials to generate stable baseline values:
• Beam walk test: Mice were trained to traverse a series of
elevated beams 7mm in diameter to reach an enclosed escape platform. Mice were placed on 1 end of the beam, and
the latency to traverse the central 80% of the beam toward
the enclosed escape platform was recorded. Motor test data
were analyzed as mean latency to cross the beam (3 trials)
compared with the internal baseline values.
• Rotor-rod test: After a 1-minute adaptation period on the
rod at rest, the rod was continuously accelerated by 40rpm
over 4 minutes, and the length of time mice remained on
the rod (fall latency) was recorded. Data were analyzed as
percentage of mean duration (3 trials) on the rotor-rod
compared with the internal baseline values.
• Corner test: The apparatus to perform the test consists of 2
boards (30 ⫻ 30cm2) attached at their edges at a 30° angle
with a small opening along the joint between the boards to
encourage entry into the corner. The mouse was placed between the 2 angled boards facing the corner and half way to
the corner. The turns in 1 versus the other direction were
recorded from 10 trials for each test. Turning movements
that were not part of a rearing movement were not scored.
Data were presented as turns to the left per test.
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FIGURE 1: Endothelial progenitor cell (EPC) culture and identification. (A) Mononuclear cells isolated from human peripheral
blood were (a) plated in fibronectin-coated wells at 5 ⴛ 105 cells/cm2. (b) After 5 days culture, the spindle-like attached
cells supposed to be EPCs were collected for use (bar ⴝ 50␮m). (B) The putative EPCs were able to (a) take up DiI-labeled
acetylated low-density lipoprotein and (b) bind the endothelial specific lectin fluorescein isothiocyanate–Ulex europeus
agglutinin-1, which were (c) colocalized in >95% cells (bar ⴝ 50␮m). (C) Immunofluorescent staining shows the putative
EPCs are positive for (a) endothelial cell markers KDR, (b) vascular endothelial cadherin (VE-Cad), (c) von Willebrand factor
(vWF), and (d) Tie-2 (bar ⴝ 50␮m). (D) The cultured cells are further confirmed by (d, e, f, j, k, l) flow cytometry analysis
after being labeled with different markers and (a, b, c, g, h, i) fluorescent-labeled immunoglobulin G with the same isotypes
for negative control. The results shows that ⬃2% of cells express (d) the hematopoietic cell marker CD34-PE, and the
majority of cells express endothelial markers (d, e) KDR (66 – 88%), (f) VE-Cad (78%), (j) CD-31 (94%), and monocyte markers
(k) CD11b (78%, k) and (l) CD14 (82%). FITC ⴝ fluorescein isothiocyanate; UEA ⴝ Ulex europeus agglutinin staining; KDR
ⴝ VEGF receptor II.
Cerebral Infarction Volume and Atrophy
Volume Determination
Serial frozen coronal sections, 20␮m in thickness and 200␮m in
interval from the frontal pole, were used, and Cresyl violet staining was used to identify the infarct area. Infarct volume (mm3)
was measured using the NIH image J. Brain atrophy volume
(mm3) was calculated by subtraction of the volume of ipsilateral
hemisphere from the volume of contralateral hemisphere.
Capillary Density and Cell Assessment
Capillary density was determined as described.18 The number of
capillaries was calculated as the mean of the vascular counts obtained from 3 pictures. The number of cells was calculated in
the same manner through a ⫻20 objective lens. Vessel counting
was conducted blinded to treatment allocation.
Statistical Analysis
Data were presented as mean ⫾ deviation. Parametric data from
the EPC-, HBEC-, and saline-treated groups were assessed using a
490
1-way analysis of variance followed by Fisher partial least-squares
difference test. A p ⬍ 0.05 was considered statistically significant.
Results
EPC Expansion and Characterization
Mononuclear cells (MNCs) from adult human blood
were cultured for 5 days in the EBM-2 medium as previously described (Fig 1).17 These putative EPCs attached
on the plate and gave rise to a spindle-like shape. Double
staining of FITC-UEA and DiI-acLDL showed that
⬎95% of these EPCs were able to uptake acLDL and
bind endothelial cell (EC)-specific lectin UEA. Based on
immunostaining, the cells expressed EC markers KDR,
VE-cadherin, vWF, and Tie-2. The phenotype of EPCs
was further examined by flow cytometry, which confirmed that the majority of the cells expressed KDR, VEcadherin, CD31, CD11b, and CD14.
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Fan et al: EPCs and Recovery
EPCs Homed to Ischemic Brain and Protected
against the Acute Ischemic Injury
DiI-EPCs were found around the microvessels in the ischemic boundary zone between microvessel walls and brain
tissue after 24 hours of tMCAO. The majority of DiIEPCs were detected in ischemia boundary zone, although
a few DiI-HBECs were found in ischemic brain after 72
hours of tMCAO (Fig 2A). The number of DiI-EPCs in
ischemic areas was higher than that of the DiI-HBECs
(30 ⫾ 6/field vs 10 ⫾ 3/field; p ⬍ 0.01; see Fig 2C),
suggesting that systemically administrated EPCs homed
into ischemic regions by a specific chemotactic mechanism rather than leaked from injured blood vessels.
To evaluate if transplanted EPCs protected against
brain injury after ischemia, 1 ⫻ 106 EPCs were systemically injected into mice. We found that infarct volume in
the EPC-treated group (13.9 ⫾ 3.3mm3) was reduced
compared with control animals (27.5 ⫾ 5.7mm3; p ⬍
0.05; see Fig 2E), suggesting that systemically delivered
EPCs not only homed into ischemic brain, but also were
able to protect against ischemic injury.
SDF-1/CXCR4 System Was Involved in EPCMediated Neuroprotection
To clarify whether SDF-1/CXCR4 was involved in EPCmediated neuroprotection, we examined CXCR4 expression in EPCs and investigated whether SDF-1 induced
EPC migration. Immunostaining detected CXCR4 expression in the majority of the cultured EPCs but not in
HBECs (Fig 3A). Flow cytometry analysis showed that
⬃80% of EPCs expressed CXCR4 (see Fig 3B). When
SDF-1 was applied into medium in the lower chamber of
the Boyden migration system, a large number of EPCs
migrated across the porous filter in a dose-dependent
manner (see Fig 3C). SDF-1–induced EPC migration was
attenuated when EPCs were pretreated with a CXCR4
inhibitor AMD3100, suggesting that SDF-1 and its receptor CXCR4 mediated EPC migration in vitro.
We tested whether SDF-1 expression was upregulated
after tMCAO. Western blot showed that SDF-1 expression
increased at 24 hours after tMCAO; such high expression
was maintained at least 7 days ( p ⬍ 0.05, Fig 4A). In addition, immunostaining showed that SDF-1–positive cells
were distributed in the ischemic perifocal region but not in
the contralateral hemisphere. Based on double staining and
cell morphology, SDF-1 was expressed in endothelial cells,
neurons, and astrocytes (see Fig 4B).
We further examined whether pretreatment of EPCs
with AMD3100 inhibited EPC-induced neuroprotection.
Infarct volume was significantly increased in the mice injected by AMD3100-pretreated EPCs, compared with the
mice treated with nonpretreated EPCs (22.8 ⫾ 4.9mm3
April, 2010
FIGURE 2: Intravenously delivered endothelial progenitor
cells (EPCs) home to ischemic brain. (A) Photomicrographs
show that DiI-labeled EPCs migrated (b, c) across microvessel
walls at 24 hours after injection and (e) into the ischemic area
after 72 hours of injection. Inserted boxes show increased
magnification of cells. In the control animals, (a) DiI-labeled
human brain endothelial cells (HBECs) do not show at 24
hours after injection, and (d) few cells are observed in the
ischemic zone. 4ⴕ,6-Diamidino-2-phenylindole was used for
the counterstaining. Bar ⴝ 50␮m. (B) The figure shows the
ischemic area and the ischemic boundary area. (C) The bar
graph shows that a great number of DiI-EPCs homed into
ischemic brains compared with DiI-HBECs 72 hours after intravenous injection. Data are mean ⴞ standard deviation
(SD), n ⴝ 6 in each group. (D) Nissl staining indicates that
EPC-treated mice have much smaller infarct areas than those
of control animals. The mice are subjected to 1 hour of middle cerebral artery occlusion followed with reperfusion and
EPC transplantation or an equal volume of saline as a control
through the left jugular vein. After 3 days of reperfusion, the
ischemic brains were collected, and brain sections were prepared. (E) A bar graph of quantitative analysis shows that
systemic administration of EPC significantly reduces the infarct volume 3 days after transient MCAO compared with
control animals. Data are mean ⴞ SD, n ⴝ 6 in each group.
*p < 0.05, EPC-treated mice versus saline-treated mice.
[Color figure can be viewed in the online issue, which is available at www.interscience.wiley.com.]
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FIGURE 3: Endothelial progenitor cell (EPC)-expressed CXCR4 and stromal-derived factor-1 (SDF-1) induced EPC migration in
vitro. (A) The photomicrographs show the expression of CXCR4, the receptor of SDF-1 (red) on adult human brain endothelial
cells (HBECs) (a) and EPCs (b), with the counterstaining of 4ⴕ,6-diamidino-2-phenylindole (blue). Few adult endothelial cells express
CXCR4, whereas the majority of EPCs do. Bar ⴝ 50␮m. (B) The scatter graph shows CXCR4 expression on EPCs detected with
the flow cytometry. (a) Control staining with immunoglobulin G with the same isotope as CXCR4 antibody. (b) More than 80%
of cultured EPCs express CXCR4. (C) Bar graphs show that SDF-1 induced EPC migration assays with the Boyden chamber
system. After 18 hours of SDF-1 stimulation, cells crossing the porous filter are counted, and data are presented as fold increase
in migrating cells relative to the control medium. (a) A large number of EPCs migrated across the porous filter compared with
control animals, whereas pretreatment with 5␮g/ml of AMD3100 significantly inhibited SDF-1–induced EPC migration (*p < 0.01,
SDF-1 treated group vs phosphate-buffered saline [PBS]-treated control group; **p < 0.01, AMD3100-treated EPC group vs
non–AMD3100-treated group). (b) The bar graph shows that even 1ng/ml of SDF-1 can significantly increase EPC migration (*p <
0.05, SDF-1–treated group vs PBS-treated control group). (c) The bar graph shows that as little as 1␮g/ml of AMD3100 significantly inhibits SDF-1–induced EPC migration (*p < 0.05, AMD3100-treated group vs nontreated group). All the results are
expressed as mean ⴞ standard deviation, and each panel represents the results of at least 3 independent experiments performed
in triplicate. VEGF ⴝ vascular endothelial growth factor. SSC-H ⴝ side scatter. [Color figure can be viewed in the online issue,
which is available at www.interscience.wiley.com.]
vs 13.9 ⫾ 3.3mm3; p ⬍ 0.05; Fig 5), suggesting that the
EPC-induced neuroprotective effect was abolished by the
blockage of CXCR4 with AMD3100.
EPCs Reduced Brain Cortex Atrophy and
Improved Long-Term Neurobehavioral
Outcome after tMCAO
We further evaluated the potential long-term neuroprotection of EPCs against ischemic brain injury. The overall atrophy volume of hemisphere in the EPC-treated mice
(18 ⫾ 4mm3) was significantly reduced compared with the
saline- (31 ⫾ 8mm3) or HBEC-treated mice (33 ⫾ 8mm3;
p ⬍ 0.05; Fig 6A). We also examined neurobehavioral
functions. Beam walk showed that tMCAO mice with
EPC treatment showed significant improvements in perfor492
mance after 7 days of tMCAO, as compared with the controls ( p ⬍ 0.05; see Fig 6B). Rotor-rod test showed that a
significant difference in motor learning was observed between EPC-treated and control mice after tMCAO.
HBEC-treated mice showed transitory improvement at 14
days of tMCAO, but reversed at 28 days of tMCAO ( p ⬍
0.05; see Fig 6B). Corner test demonstrated that EPCtreated mice reduced preferential turning compared with
control animals at 7, 14, and 28 days after tMCAO.
EPCs Transplantation Promoted Angiogenesis
in Ischemic Brain
EPCs can secret various angiogenic growth factors to
promote angiogenesis.19 Capillary density was higher in
the mice treated with EPCs (261 ⫾ 53/field) compared
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Discussion
FIGURE 4: Stromal-derived factor-1 (SDF-1) expression was
upregulated in mouse brain subjected to transient middle
cerebral artery occlusion (tMCAO) after reperfusion. (A)
The Western blot results show SDF-1 expression in ischemic brains, with ␤-actin as an internal control (upper
panel). SDF-1 expression significantly increases at 1 day, 3
days, and 7 days, with a peak at 1 day after tMCAO
(lower panel). Data are mean ⴞ standard deviation, n ⴝ 6.
*p < 0.05, SDF-1 expression in the brains of tMCAO mice
versus saline-treated mice. (B) The photomicrographs show
the immunofluorescent staining of SDF-1 (green) in (a) nonischemic brains and (b) brains subjected to 1 hour of tMCAO with 24 hours of reperfusion. Double staining (c) of
SDF-1 (green) with CD31 (red, upper panel) or glial fibrillary acidic protein (red, middle panel) shows that SDF-1 is
expressed in endothelial cells and astrocytes. Bar ⴝ 50␮m.
Based on the cell morphology, SDF-1 is also expressed in
neurons (c, lower panel). Bar ⴝ 50␮m in (B) and Bar ⴝ
20␮m in (Ca,Cb,Cc). [Color figure can be viewed in the
online issue, which is available at www.interscience.wiley.
com.]
with the saline (115 ⫾ 25/field) or HBEC (118 ⫾ 28/
field; p ⬍ 0.01; Fig 7A) groups. Double immunostaining showed that many bromodeoxyuridine-positive cells
were colocalized with CD31-positive cells in the mouse
brain that received EPC transplantation (see Fig 7B). An
antihuman CD34 antibody, which does not cross-react
with mouse ECs, was used to track the transplanted
hEPCs. We found several human CD34-positive microvessels in ischemic brains (see Fig 7C). These results
provided the evidence that transplanted EPCs induced
microvessel formation not only by promoting angiogenesis, but also by initiating de novo vascularization in
ischemic brain.
April, 2010
In the present study, we determined that the delivered
hEPCs were able to home to ischemic brain as early as 24
hours after delivery, which not only protected against
ischemic brain injury at acute stage, but promoted brain
regeneration by promoting angiogenesis, eventually leading to improved neurobehavioral outcomes. Our data also
showed that blocking CXCR4 on EPCs abolished the
EPC-induced neuroprotection, suggesting that SDF-1/
CXCR4 played a crucial role in EPC-mediated brain protection. Our findings provided evidence that EPC-based
cell therapy was a novel strategy for ischemic stroke
therapy.
We isolated and enriched EPCs by cultivating
MNCs collected from circulating blood. Cultureexpanded EPCs produced better revascularization than
fresh-isolated CD34-positive cells after translation.20,21
The advantage of MNC cultivation for cell therapy is that
it gives rise to both EPCs and angiogenic cells derived
from different cell linage origins. In general, EPCs, especially ex vivo cultured ones, represented heterogeneous
groups without a straightforward definitive marker. Only
a small percentage of cells that adhered early in culture
were “true EPCs,” which stayed quiescent at the early
FIGURE 5: AMD3100-treated endothelial progenitor cells
(EPCs) abolished cerebral protection against ischemic injury. (A) Nissl staining shows that the infarct areas in mice
with AMD3100-treated EPC was much bigger than in those
treated with EPCs. The mice were subjected to 1 hour of
MCAO followed with reperfusion and transplantation of
EPCs or 5␮g/ml of AMD3100-treated EPCs through the
left jugular vein. After 3 days of reperfusion, the ischemic
brains were collected, and brain sections were prepared.
(B) A bar graph shows that the brain infarction volume of
mice with AMD3100-treated EPCs significantly increased
compared with the nontreated EPC group. Data are
mean ⴞ SD, n ⴝ 6. *p < 0.05, AMD3100-treated EPC
group versus nontreated EPC group. [Color figure can be
viewed in the online issue, which is available at www.
interscience.wiley.com.]
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FIGURE 6: Endothelial progenitor cells (EPC) transplantation reduced mouse cortical atrophy and improved neurobehavioral
outcomes. (A) EPC-treated mice showed reduced cortical atrophy after transient middle cerebral artery occlusion (tMCAO)/
reperfusion. The mice were subjected to 1 hour of MCAO followed with reperfusion and EPC transplantation. Saline and
human brain endothelial cells (HBECs) were used as controls. The ischemic brains were collected 4 weeks after tMCAO. (a)
Nissl staining shows much smaller atrophy in the brains of the EPC-treated group than those of the saline- and HBECtreated groups. (b) Quantitative analysis shows that systemic administration of EPCs significantly reduced the brain atrophy
4 weeks after tMCAO compared with the saline- and HBEC-treated groups. Data are mean ⴞ standard deviation (SD), n ⴝ
6 in each group. *p < 0.05, EPC-treated mice versus saline- or HBEC-treated mice. (B) EPC-treated mice show improved
neurobehavioral outcome. All mice are trained for 3 days before tMCAO with 3 consecutive trials to generate stable
baseline values. Neurobehavioral functions of mice are tested at 1 day, 7 days, 14 days, and 28 days after tMCAO. Line
graphs show that EPC-treated mice showed significantly improved performance in crossing the beam walk (a, beam test),
staying on the rotor-rod (b, rotor-rod test [Rotrod]), and turning preferentially toward the nonimpaired ipsilateral side (c,
corner test) compared with mice treated with saline or HBEC after tMCAO. Data are mean ⴞ SD, n ⴝ 6 in each group. *p <
0.05, EPC-treated mice versus saline- or HBEC-treated mice. [Color figure can be viewed in the online issue, which is
available at www.interscience.wiley.com.]
stage of culture but quickly proliferated and differentiated
to late EPCs or ECs after 2 weeks of culture.22 Interestingly, we found several human-specific CD34-positive
microvessels in mouse brains after 4 weeks of EPC transplantation, which could be differentiated from transplanted hEPCs.
Although innate EPCs could be recruited into peripheral blood after injury, they may not provide sufficient supply for severe injuries. Moreover, EPCs in the
peripheral blood decreased in patients with ischemic
stroke. Therefore, the rationale of EPC therapy in brain
ischemia was to transplant exogenous EPCs to provide
494
sufficient EPCs to participate in brain repairing. Previous
reports showed that systemically injected EPCs were able
to home to local injured tissue, promote vascular remodeling and revascularization, and improve tissue repair and
regeneration.23 Animal studies showed that CD34positive cells or EPCs derived from BM reduced ischemic
brain injury.16,24,25 We observed that intravenously transplanted EPCs homed into the ischemic zone as early as 24
hours after tMCAO, which not only significantly reduced
brain infarct volume at the acute stage, but decreased cortical atrophy and improved long-term neurobehavioral
outcomes. When homing to the ischemic zone, the transVolume 67, No. 4
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Š
planted EPCs secreted various growth factors, such as insulinlike growth factor-1, vascular endothelial growth factor, granulocyte-macrophage colony-stimulating factor,
granulocyte colony-stimulating factor, and SDF-1.14,19 In
this study, EPC-mediated neuroprotection at the acute
phase of ischemic brain injury was evaluated by measuring
infarct volume. EPC-induced neuroprotection at the
chronic phase of brain injury was evaluated by assessing
the degree of brain cortical atrophy and neuronal behavioral tests.7,24,26 Brain atrophy happened after tMCAO at
the subacute phase. Therefore, Nonaka et al measured the
atrophy volume at 7, 35, and 90 days after tMCAO.26
The effect at the early stage of ischemia may result from
neuroprotective growth factors, inhibiting cell apoptosis
or promoting the neuronal survival. On the other hand,
these EPC-secreted growth factors could induce local angiogenesis and recruit endogenous progenitor cells for
neurogenesis.27–29 In addition, transplanted EPCs induced de novo angiogenesis, as human CD34-positive microvessels were found in the ischemic brain with EPC
April, 2010
FIGURE 7: Intravenously delivered endothelial progenitor
cells (EPCs) promoted local angiogenesis in ischemic brain.
(A) CD31 immunostaining shows microvessels in mouse brain
treated with (a) saline, (b) human brain endothelial cells
(HBECs), and (d) EPCs after transient middle cerebral artery
occlusion. (c) The ischemic area and the ischemic boundary
area are shown. The bar graph (e) shows that the number of
microvessels in EPC-treated mice was significantly increased
compared with that in saline- or HBEC-treated mice. Data are
mean ⴞ standard deviation, n ⴝ 6 in each group. *p < 0.05,
the microvessel number in EPC-treated mice versus that of
HBEC- or saline-treated mice. Bar ⴝ 100␮m. (B) Photomicrographs show (a, b, c) CD31 (red) and (d, e, f) bromodeoxyuridine (green) double staining in ischemic brain of (a, d, g)
saline-, (b, e, h) HBEC-, and (c, f, i) EPC-treated mice. (g, h, i)
In the EPC-treated group, increased bromodeoxyuridinepositive cells were localized in endothelial cells (yellow), suggesting that EPC transplantation promotes new vessel formation in ischemic brain. Bar ⴝ 50␮m. Inserted boxes show
increased magnification of cells. (C) Photomicrographs of human CD34-positive microvessels in ischemic boundary areas
of mouse brain. Antihuman CD34 antibody was used to track
the transplanted human EPCs in mouse brain. The human
CD34-positive microvessels were not observed in the (a)
saline- or (b) HBEC-treated groups, but formed in (c) EPCtransplanted mouse brain. Bar ⴝ 100␮m. [Color figure can be
viewed in the online issue, which is available at www.interscience.wiley.com.]
transplantation. Therefore, the transplanted EPCs after
angiogenesis and de novo vascularization increased microvessel numbers and promoted revascularization, which
enhanced local blood flow, improved cell survival, and
promoted brain regeneration. Furthermore, implanted circulating CD34-positive cells may differentiate into glial
and neurons, thereby enhancing neuroplastic effects in the
ischemic brain.25
CXCR4 was considered as a unique receptor of
SDF-1, and AMD3100 was used as a specific inhibitor of
CXCR4. However, SDF-1 may bind CXCR7 as a second
receptor.30 It was documented that SDF-1/CXCR4 signaling played a primary role in governing stem cell homing and engraftment in the BM,31 but the role of CXCR7
was still unknown. SDF-1 was reported to mediate BM
cell homing to brain ischemic regions.32 However, other
studies demonstrated that blockage of CXCR4 did not
inhibit BM cells homing in the recipient mice after transplantation, whereas blockage of both integrin ␣4/vascular
cell adhesion molecule-1 and SDF-1/CXCR4 did.33
Overexpression of SDF-1 by gene transfer in the heart
after myocardial infarction increased BM cell recruitment
but did not enhance recruitment into the heart without
infarction,34 suggesting that a more complicated mechanism was involved in SDF-1 function in stem cell homing. To investigate if the SDF-1/CXCR4 system was involved in EPC-mediated neuroprotection, we analyzed
SDF-1 expression in ischemic brain and CXCR4 expression on EPCs. Consistent with others’ reports, we found
495
ANNALS
of Neurology
that SDF-1 expression was increased in ischemic brains
starting 24 hours after tMCAO, by neurons, astrocytes,
and ECs as expected. In vitro studies showed that EPCexpressed CXCR4 and SDF-1 was capable of inducing
EPC migration. Such migration was robustly inhibited
when EPCs were pretreated with AMD3100. In vivo
studies also showed that EPC treatment could protect
against ischemic brain injury, but AMD3100-pretreated
EPCs could not. It has been reported that preincubation
of transplanted EPCs with antibodies against CXCR4 reduced EPC incorporation, and nude CXCR4⫹/⫺ mice
showed impaired capacity to enhance recovery of ischemic
blood flow.35,36 Taken together, it was suggested that
SDF-1/CXCR4 played a critical role in EPC migration
and EPC-mediated neuronal protection after ischemia.
However, we cannot address the question of whether
CXCR7, the alternative receptor of SDF-1, is involved in
SDF-1 mediated neuroprotection. It was reported that
CXCR7 is involved in SDF-1-mediated G protein signaling by heterodimerizing with CXCR4.37 It has been reported that AMD3100 not only binds and blocks
CXCR4 as an inhibitor, but also binds and activates
CXCR7 as an allosteric agonist of CXCR7.38 CXCR7
scarcely expressed on CD34⫹ progenitors and CXCR7
blockers did not affect SDF-1/CXCR4 signaling at early
stages such as Akt signaling as well as CXCR4-mediated
chemotaxis, but attenuated the CXCR4 rearrangement after binding SDF-1.39 This study did not explore interaction between SDF-1 and CXCR7. The potential role
of CXCR7 in ischemic brain injury needs further investigation.
SDF-1 is regulated by hypoxia-inducible factor-1
(HIF-1).35,40 Ischemia and alteration of HIF-1 lead to increasing SDF-1 expression, which cooperates with adhesion molecules such as vascular cell adhesion molecule-1
and intercellular adhesion molecule-141 to trigger the recruitment of CXCR4-positive EPCs into injured brain regions. EPCs either secreted various growth factors to drive
angiogenesis/neovascularization or were incorporated into
newly formed vessels, accelerating the revascularization. It
was reported that binding of CXCR4 with SDF-1␣ activated the PI3K/Akt/eNOS signaling pathway and decreased EPC apoptosis under serum deprivation. Pretreatment of EPCs with SDF-1 increased their proangiogenic
potential by upregulating the expression of integrin ␣4
and ␣M subunits and enhancing the EPC adhesion to
activated endothelium.42
We used hEPCs in this study with the vision of developing clinical ischemic stroke therapy using hEPC
transplantation. To avoid the xenograft rejection, we used
immune-deficient nude mice instead of wide-type mice.
496
Despite the potentially different response to infarct from
wide-type mice, the use of control groups helped to confirm the brain protection effect of EPCs. Nude mice have
been widely used in similar research.7,36,43 Our studies
provide evidence for the neuroprotection of EPCs in ischemic stroke. Further studies are needed to elucidate the
mechanism of such protection and to optimize the dose
and timing of EPC transplantation.
Acknowledgment
This study was supported by the NIH NINDS (grants
R01 NS27713, W.L.Y.; R21 NS50668, G.-Y.Y.; and P01
NS044145, W.L.Y., G.-Y.Y.). We thank V. Gungab for
editorial assistance and the staff of the Center for Cerebrovascular Research at University of California, San
Francisco for collaborative support.
Potential Conflicts of Interest
Nothing to report.
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