TISSUE-SPECIFIC STEM CELLS

Nitric Oxide Donor Upregulation of Stromal Cell-Derived Factor-1/

Chemokine (CXC Motif) Receptor 4 Enhances Bone Marrow
Stromal Cell Migration into Ischemic Brain After Stroke
XU CUI,a JIELI CHEN,a ALEX ZACHAREK,a YI LI,a CYNTHIA ROBERTS,a ALISSA KAPKE,b
SMITA SAVANT-BHONSALE,c MICHAEL CHOPPa,d
Departments of aNeurology and bBiostatistics and Research Epidemiology, Henry Ford Health Sciences Center,
Detroit, Michigan, USA; cDepartment of Neurobiology, Theradigm, Inc., Baltimore, Maryland, USA;
d
Department of Physics, Oakland University, Rochester, Michigan, USA
Key Words. DETA-NONOate • Stromal cell-derived factor-1/chemokine receptor 4 • Matrix metalloproteinases
Bone marrow stromal cell • Migration • Stroke

ABSTRACT
Stromal cell-derived factor-1 (SDF1) and its chemokine
(CXC motif) receptor 4 (CXCR4), along with matrix metal-
loproteinases (MMPs), regulate bone marrow stromal cell
(BMSC) migration. We tested the hypothesis that a nitric
oxide donor, DETA-NONOate, increases endogenous isch-
emic brain SDF1 and BMSC CXCR4 and MMP9 expres-
sion, which promotes BMSC migration into ischemic brain
and thereby enhances functional outcome after stroke.
C57BL/6J mice were subjected to middle cerebral artery
occlusion (MCAo), and 24 hours later, the following were
intravenously administered (n ⴝ 9 mice per group): (a)
phosphate-buffered saline; (b) BMSCs (5 ⴛ 105); (c) 0.4
mg/kg DETA-NONOate; (d) combination of CXCR4-inhibi-
tion BMSCs with DETA-NONOate; and (e) combination of
BMSCs with DETA-NONOate. To elucidate the mecha-
nisms underlying combination-enhanced BMSC migration,
transwell cocultures of BMSC with mouse brain endothe-
lial cells (MBECs) or astrocytes were performed. Combi-
nation treatment significantly improved functional out-
Disclosure of potential conflicts of interest is found at the end of this article.
come after stroke compared with BMSC monotherapy
and MCAo control, and it increased SDF1 expression in
the ischemic brain compared with DETA-NONOate
monotherapy and MCAo control. The number of BMSCs
in the ischemic brain was significantly increased after
combination BMSC with DETA-NONOate treatment
compared with monotherapy with BMSCs. The number
of engrafted BMSCs was significantly correlated with
functional outcome after stroke. DETA-NONOate signif-
icantly increased BMSC CXCR4 and MMP9 expression
and promoted BMSC adhesion and migration to MBECs
and astrocytes compared with nontreatment BMSCs. In-
hibition of CXCR4 or MMPs in BMSCs significantly
decreased DETA-NONOate-induced BMSC adhesion and
migration. Our data demonstrate that DETA-NONOate
enhanced the therapeutic potency of BMSCs, possibly via
upregulation of SDF1/CXCR4 and MMP pathways, and
increased BMSC engraftment into the ischemic brain.
STEM CELLS 2007;25:2777–2785

receptor [5] chemokine (CXC motif) receptor 4 (CXCR4) reg-

INTRODUCTION
The regenerative potential of bone marrow stromal cells
(BMSCs) has been demonstrated in myocardial, limb, and brain
ischemia [1]. Systemically administered BMSCs selectively mi-
grate into the injury site [2] and dose-dependently improve
neurological functional recovery after stroke [3]. The effect
of BMSC transplantation is dependent on the number of en-
grafted BMSCs [3]. In addition, the success of a vascular route
for BMSC treatment has been limited by the low migration
efficiency of the transplanted BMSCs into the lesioned area [4].
Thus, priming the ischemic brain to promote BMSC migration
into the ischemic brain may augment BMSC regenerative treat-
ment of stroke.
Chemokines are important factors controlling cellular mi-
gration. Stromal cell-derived factor-1 (SDF1) and its unique
ulate the tracking of various types CXCR4-positive cells [6].
BMSCs can be efficiently transduced to express CXCR4, and
transduced BMSCs migrate rapidly toward SDF1␣ [7]. Intrace-
rebral injection of recombinant human SDF1␣ stimulates the
homing of transplanted BMSCs to the site of injection in the brain
[8]. Nitric oxide (NO) donors significantly enhance the SDF1␣-
induced cell migration [9]. Intravenous infusion of endothelial
nitric oxide synthase (eNOS)⫺/⫺ bone marrow (BM) cells into
wild-type mice decreases BM migration into lesion area [10].
Thus, NO-mediated SDF1/CXCR4 likely controls the traffick-
ing of transplanted BMSCs. In addition, matrix metalloprotein-
ases (MMPs) are involved in SDF1/CXCR4-induced chemotaxis
of human hematopoietic progenitor cells across subendothelial
basement membranes [11]. SDF1␣ gradient increases the chemo-
taxis of bone marrow and blood CD34(⫹) cells, which is blocked
by inhibitors of MMPs [11].

Correspondence: Michael Chopp, Ph.D., Neurology Research, E&R Building, Room 3056, Henry Ford Hospital, 2799 West Grand
Boulevard, Detroit, Michigan 48202, USA. Telephone: 313-916-3936; Fax: 313-916-1318; e-mail: chopp@neuro.hfh.edu Received March
9, 2007; accepted for publication July 12, 2007; first published online in STEM CELLS EXPRESS July 19, 2007. ©AlphaMed Press
1066-5099/2007/$30.00/0 doi: 10.1634/stemcells.2007-0169

STEM CELLS 2007;25:2777–2785 www.StemCells.com

2778
DETA-NONOate ([Z]-1-[N-(2-aminoethyl)-N-(2-ammonio-
ethyl) aminio] diazen-1-ium-1,2-diolate), a NO donor, promotes
angiogenesis and improves neurological outcome after stroke
[12]. Our previous studies have shown that the combination of
a subtherapeutic dose of DETA-NONOate and BMSCs acts
additively to improve the therapeutic outcome after stroke in
rats [13]. However, the mechanism of combination treatment-
induced additive functional improvement after stroke has not
been investigated. In this study, we sought to test the mechanism
by which combination treatment of BMSC with DETA-NONO-
ate amplified restorative therapy after stroke in mice. We hy-
pothesized that DETA-NONOate treatment promotes BMSC
migration into the ischemic brain by increasing SDF1 within the
compromised brain.
MATERIALS AND METHODS
All experiments were conducted in accordance with the standards
and procedures of the American Council on Animal Care and the
Henry Ford Health System Institutional Animal Care and Use
Committee.
BMSC Culture and Labeling with
5-Bromo-2ⴕ-Deoxyuridine
Mouse BMSCs (M-126, P9; Cognate BioServices, Inc., Baltimore,
MD, http://www.cognatetherapeutics.com) were incubated in HyQ
MEM Alpha Modification (HyClone, Logan, UT, http://www.
hyclone.com) with 20% fetal bovine serum (Gibco, Grand Island,
NY, http://www.invitrogen.com) and 1% antibiotins (penicillin-
streptomycin; Gibco) and maintained at 37°C in 5% CO2/95%
ambient mixed air. For injection to animals, BMSCs were labeled
with 5-bromo-2⬘-deoxyuridine (BrdU) (30 ␮g/ml; Sigma-Aldrich,
St. Louis, http://www.sigmaaldrich.com) in vitro for 3 days [3].
Passage 3–5 BMSCs were used.
Middle Cerebral Artery Occlusion Model and
Experimental Groups
Adult male C57BL/6J mice (22–25 g; Charles River Laboratories,
Wilmington, MA, http://www.criver.com) were subjected to tran-
sient (2.5 hours) monofilament right middle cerebral artery occlu-
sion (MCAo) [14]. Mice were randomly divided into five groups
(n ⫽ 9 mice per group) 24 hours after MCAo, and the following was
injected via the tail vein: (a) 0.2 ml of phosphate-buffered saline
(PBS) for control; (b) 5 ⫻ 105 BMSCs; (c) DETA-NONOate (0.4
mg/kg in 0.2 ml of PBS; Alexis Biochemical, San Diego, http://
www.alexis-corp.com); (d) combination of BMSCs (5 ⫻ 105) pre-
treated with specific CXCR4 inhibitor AMD3100 [15] (20 ␮M for
3 hours) with DETA-NONOate (0.4 mg/kg); or (e) combination of
BMSCs (5 ⫻ 105) with DETA-NONOate (0.4 mg/kg). In an earlier
study, a single dose of 0.4 mg/kg DETA-NONOate did not improve
functional outcome after stroke [13]. In addition, we have shown
that a dose of 5 ⫻ 105 BMSCs does not provide a significant benefit
on functional outcome after stroke (J. Chen, C.L. Zhang, and M.
Chopp, unpublished data). In this study, we investigated whether a
subtherapeutic dose of DETA-NONOate and a subtherapeutic dose
of BMSCs induce additive or superadditive effects. Therefore, we
selected subtherapeutic doses of 0.4 mg/kg DETA-NONOate (sin-
gle injection) and 5 ⫻ 105 BMSC.
Behavioral Tests
A modified neurological severity score (mNSS) [14] and foot-fault
tests [14] were performed by a blinded investigator before MCAo
and at 1, 7, and 14 days after MCAo, as previously described [14].
Histological and Immunohistochemistry Assessment
Mice were sacrificed at 14 days after MCAo. The brains were fixed
by transcardial perfusion with saline, followed by perfusion and
immersion in 4% paraformaldehyde, before being embedded in
DETA-NONOate Enhances BMSC Migration After Stroke
paraffin [14]. A standard paraffin block was obtained from the
center of the lesion (bregma, ⫺1 mm to ⫹1 mm). A series of
6-␮m-thick sections was cut from the block. Every 10th coronal
section for a total five sections was used for immunohistochemical
staining. Antibody against BrdU (1:100; Roche, Indianapolis, http://
www.roche-applied-science.com) and SDF1 (1:250; Santa Cruz
Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) single
immunostaining was performed to detect the number of engrafted
BMSCs and the expression of SDF1 in the ischemic brain, respec-
tively. Control experiments consisted of staining brain coronal
tissue sections as outlined above, but the primary antibodies were
omitted, as previously described [16].
Double Immunofluorescence Staining
To identify SDF1-reactive cells colocalized with brain endothelial
cells or astrocytes, double immunofluorescence staining was used.
von Willebrand factor (vWF) is a marker for endothelial cells. Glial
fibrillary acidic protein (GFAP) is a marker for astrocytes. Double
immunofluorescence labeling for SDF1 with vWF and SDF1 with
GFAP was performed. Each coronal section was first treated with
the primary anti-SDF1 antibody with fluorescein isothiocyanate
(FITC), followed by anti-vWF (1:400; DAKO, Carpinteria, CA,
http://www.dako.com) or anti-GFAP (1:1,000; DAKO) with Cy3
staining. Control experiments consisted of staining brain coronal
tissue sections as outlined above, but the primary antibodies were
omitted, as previously described [16]. SDF1 and GFAP double
immunofluorescent images were acquired using fluorescent micros-
copy (Axiophot2, HB0100 W/2; Carl Zeiss, New York, http://
www.zeiss.com) with a digital camera (C4742-95; Hamamatsu,
Hamamatsu City, Japan, http://www.hamamatsu.com). SDF1 and
vWF double immunostaining confocal images were acquired using
an MRC 1024 (argon and krypton; Bio-Rad, Hercules, CA, http://
www.bio-rad.com) laser-scanning confocal imaging system
mounted onto a Carl Zeiss microscope, as previously described
[17].
Quantification
For measurement of the numbers of engrafted BMSCs, the total
numbers of BrdU-labeled BMSCs in both the ipsilateral and con-
tralateral hemispheres were counted. For quantification, the percent-
age of the SDF1-positive area was measured in the ischemic border
area [18].
BMSC Culture
To test whether DETA-NONOate regulates BMSC CXCR4 and
MMP9 expression, BMSCs were cultured and treated with (a)
nontreatment control or (b) 0.4 ␮M DETA-NONOate. For real-time
polymerase chain reaction (PCR), one set of the experimental group
was terminated at 3 hours after treatment. For immunostaining,
zymography, and flow cytometric analysis (fluorescence-activated
cell sorting [FACS]), three additional sets of experimental groups
were terminated at 24 hours after treatment.
Real-Time PCR
Brain tissue (ischemic boundary zone) from the MCAo control and
DETA-NONOate monotherapy groups, and cultured BMSCs were
collected, and total RNA was isolated with TRIzol (Invitrogen,
Carlsbad, CA, http://www.invitrogen.com) following a standard
protocol [19]. Quantitative PCR was performed using the SYBR
Green real-time PCR method on an ABI 7000 PCR instrument (Ap-
plied Biosystems, Foster City, CA, http://www.appliedbiosystems.
com) using three-stage program parameters provided by the manu-
facturer. Each sample was tested in triplicate, and relative gene
expression data were analyzed using the 2⫺⌬⌬CT method. The
following primers for real-time PCR were designed using Primer
Express software (Applied Biosystems): GAPDH (forward, AGA
ACA TCA TCC CTG CAT CC; reverse, CAC ATT GGG GGT
AGG AAC AC), CXCR4 (forward, GGC TGT AGA GCG ATG
TTT GC; reverse, GTA GAG GTT GAC AGT GTA), and MMP9
(forward, AAT CTC TTC TAG AGA CTG GGA AGG AG; re-
verse, AGC TGA TTG ACT AAA GTA GCT GGA).
Cui, Chen, Zacharek et al.
SDS-Polyacrylamide Gel Electrophoresis
Zymography
Conditioned media were collected and concentrated using a Centri-
con concentrator (Millipore, Bedford, MA, http://www.millipore.
com). Protein concentrations were analyzed with a Bio-Rad system.
Equal amounts of protein (20 ␮g/lane) for each sample were mixed
with 2⫻ sample buffer and loaded on a 10% polyacrylamide gel
incorporated with 0.1% gelatin for electrophoresis. MMP9 zymo-
graphic standards were used as positive controls (Chemicon, Te-
mecula, CA, http://www.chemicon.com). After electrophoresis, gels
were washed in 2.5% Triton X-100 for 1 hour, incubated for 18
hours at 37°C in collagenase buffer, and stained for 1 hour with
0.1% Coomassie Brilliant Blue. Gelatinolytic activity was visual-
ized as a transparent band against a blue background. Zymography
was measured for quantification analysis by spot density measure-
ment using a digital imaging analysis system (Alpha Innotech,
Mount Prospect, IL, http://www.alphainnotech.com) [20, 21].
Immunohistochemistry
Antibody against CXCR4 (1:400; Chemicon) and MMP9 (1:200;
Santa Cruz Biotechnology) conjugated with Cy3 (1:200; Jackson
Immunoresearch Laboratories, West Grove, PA, http://www.
jacksonimmuno.com) immunostaining was performed. Nuclei were
identified by 4⬘,6-diamidino-2-phenylindole dihydrochloride (1:10,000;
Molecular Probes Inc., Eugene, OR, http://probes.invitrogen.com).
The percentage of CXCR4- and MMP9-positive cells was measured
using MCID software (Imaging Research, Saint Catharines, ON,
Canada, http://www.imagingresearch.com).
Flow Cytometric Analysis of CXCR4 Expression
Single-cell suspensions (1 ⫻ 106) were incubated, fixed with 4%
paraformaldehyde, and rinsed twice with 1⫻ PBS with CXCR4
antibody (1:250; AB1846; Chemicon) conjugated with FITC (1:
200; Jackson Immunoresearch Laboratories). Immunostaining anal-
ysis was performed on a BD FACSCalibur flow cytometry (BD
Biosciences, San Diego, http://www.bdbiosciences.com). At least
104 cells were analyzed in all specimens. The percentage of
CXCR4-positive cells was determined using CellQuest software
(Becton Dickinson Canada Inc., Oakville, ON, Canada, http://www.
bd.com).
BMSC Adhesion
To test whether DETA-NONOate increases BMSC adhesion to
brain endothelial cells and astrocytes, and to investigate the signal-
ing pathway of DETA-NONOate upregulation of BMSC adhesion,
green fluorescent protein (GFP)-BMSCs were cultured with mouse
brain endothelial cells (MBECs) (American Type Culture Collec-
tion, Manassas, VA, http://www.atcc.org) and astrocytes (American
Type Culture Collection). MBECs or astrocytes (1 ⫻ 104 cells per
well) were placed in 96-well chambers and pretreated with or
without DETA-NONOate (0.4 ␮M) for 24 hours, separately. Then,
BMSCs were added to MBECs or astrocytes and treated with or
without AMD3100 (20 ␮M; AnorMED, Groton, CT, http://www.
anormed.com), a specific antagonist of CXCR4, or GM6001 (10
␮M; Chemicon), a selective inhibitor of MMPs (MMP1, 2, 3, 8, and
9), for an additional 3 hours in serum-starved Dulbecco’s modified
Eagle’s medium (n ⫽ 6 wells per group) [22]. The numbers of
adherent GFP-BMSCs to MBECs or astrocytes were counted using
fluorescence microscopy (BH-2; Olympus, Tokyo, http://www.
olympus-global.com).
BMSC Migration
To test whether SDF1 and DETA-NONOate regulates BMSC
migration, GFP-BMSCs were placed in the upper chamber of a
Transwell system (Corning Life Sciences, Acton, MA, http://www.
corning.com/lifesciences) and treated with or without SDF1␣ (200
ng/ml; Sigma-Aldrich) or DETA-NONOate (0.4 ␮M) for 24 hours.
The number of BMSCs migrating to the lower chamber was
counted.
To further test whether DETA-NONOate promotes BMSC mi-
gration toward MBECs or astrocytes, transwell coculture of GFP-
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2779
BMSCs with MBECs or GFP-BMSCs with astrocytes was per-
formed. MBECs or astrocytes (5 ⫻ 104 cells per well) were placed
in the lower chamber. GFP-BMSCs were placed in a six-well
chamber. GFP-BMSCs, MBECs, and astrocytes were pretreated
with or without DETA-NONOate (0.4 ␮M) for 24 hours, separately.
In addition, 24-well Transwell polycarbonate inserts with a pore
size of 8 ␮m (Corning Life Sciences) coated with 50 ␮g/ml fi-
bronectin (Chemicon) and 0.1% gelatin were used. GFP-BMSCs
(5 ⫻ 104 cells per well) were placed in the upper chamber and
cocultured with MBECs or astrocytes for 5 hours. GFP-BMSC
migration to the lower side of the insert was counted using MCID
software [23, 24].
To test whether CXCR4 and MMPs regulate BMSC migration,
in separate experiments, MBECs or astrocytes (5 ⫻ 104 cells per
well) were placed in the lower chamber and pretreated with or
without DETA-NONOate (0.4 ␮M) for 24 hours. GFP-BMSCs
were pretreated with or without AMD3100 (20 ␮M) or GM6001 (10
␮M) to block CXCR4 or MMP, respectively, for 3 hours. Then,
GFP-BMSCs were placed in the upper chamber and cocultured with
MBECs or astrocytes for 5 hours. GFP-BMSC migration to the
lower side of the insert was then counted.
Statistical Analysis
Analysis of variance (ANOVA) was performed to compare the
functional results, SDF1 immunostaining-positive areas, and BrdU-
positive numbers in the brain tissues. A two-independent-sample t
test was used to compare CXCR4- or MMP9-reactive cell numbers
and CXCR4, MMP9, and brain tissue SDF1 mRNA levels. For cell
adhesion and migration data, ANOVAs followed by pairwise com-
parisons were performed if the overall treatment effect was signif-
icant at p ⬍ .05. Running regression models (bivariate correlation)
was used to analyze the correlation of neurological functional
outcome with the presence of BMSC number in the ischemic brain.
All data are presented as mean ⫾ SE; p ⬍ .05 is considered
significant.
RESULTS
Combination Treatment with DETA-NONOate and
BMSCs Increases the Numbers of BMSCs in the
Ischemic Brain and Improves Neurological Function
After Stroke
To determine whether DETA-NONOate and BMSC combina-
tion treatment of stroke improves neurological functional recov-
ery, a battery of functional tests were performed. Figure 1A and
1B shows that combination DETA-NONOate with BMSCs sig-
nificantly improved the functional outcome (Fig. 1A, mNSS;
Fig. 1B: foot-fault test) compared with MCAo control group at
7 and 14 days (p ⬍ .05), respectively, and compared with the
BMSC monotherapy group at 14 days in mNSS test (p ⬍ .05)
after stroke. There was no significant functional improvement
(mNSS and foot-fault tests) in AMD3100-exposed BMSC com-
bination with DETA-NONOate treatment group compared with
MCAo control.
To test whether DETA-NONOate increases BMSC delivery
into the ischemic brain, BMSCs identified by BrdU immuno-
staining were measured. BrdU-positive cell number was counted
in the ipsilateral and contralateral hemispheres, respectively.
Figure 1 shows that BrdU-positive BMSC number significantly
increased in the combination treatment group (Fig. 1E, 1F)
compared with the BMSC monotherapy group in both ipsilateral
and contralateral hemispheres (Fig. 1C, 1F, p ⬍ .05). However,
the number of BrdU-positive BMSCs did not show significantly
increase in the AMD3100-exposed BMSC combination with
DETA-NONOate treatment group compared with the BMSC
monotherapy group (Fig. 1D, 1F). Using the running regression
models to analyze the correlation of neurological functional
outcome with the presence of BMSC number in the ischemic
2780 DETA-NONOate Enhances BMSC Migration After Stroke

Figure 1. Combination DETA-NONOate with BMSC treatment of stroke increases BMSC engraftment into the ischemic brain and improves
functional recovery in mice after stroke. (A): mNSS test. (B): Foot-fault test. (C–E): H&E staining showing immunostaining for BrdU-positive
BMSCs in BMSC-alone treatment ([C], arrow), combination treatment with chemokine (CXC motif) receptor 4-inhibition BMSCs ([D], arrow), and
combination treatment with normal BMSCs ([E], arrow). (F): Quantitative data of BrdU-immunoreactive BMSCs. Scale bar ⫽ 50 ␮m. n ⫽ 9 mice
per group. Abbreviations: BMSC, bone marrow stromal cell; BrdU, 5-bromo-2⬘-deoxyuridine; MCAo, middle cerebral artery occlusion; mNSS,
modified neurological severity score.

brain, the data show that the number of BrdU-positive cells in
the ischemic brain was significantly (p ⬍ .05) and negatively
correlated with mNSS (r ⫽ ⫺0.721) and foot-fault (r ⫽
⫺0.713) values. These data suggest that increased BMSCs
present in the ischemic brain correlate with functional recovery
after stroke.
Double immunofluorescent staining showed that SDF1 pri-
marily colocalized with GFAP-stained astrocytes (Fig. 2G–
2I). Confocal images showed that SDF1 partially colocalized
with vWF-stained endothelial cells in the ischemic boundary
zone (Fig. 2K–2M).

Combination Treatment with DETA-NONOate and
BMSCs Promotes SDF1 Expression in the Ischemic
Boundary Zone
The SDF1/CXCR4 axis regulates BMSC recruitment to le-
sioned area [25]. To test whether DETA-NONOate enhance-
ment of BMSC recruitment into the ischemic brain occurs via
upregulation of the SDF1/CXCR4 pathway, SDF1 mRNA
and protein expression were measured using real-time PCR
and tissue immunostaining, respectively. Figure 2 shows that
SDF1 mRNA level significantly increased in the ischemic
boundary zone in DETA-NONOate treatment group com-
pared with MCAo control group at 14 days after stroke (p ⬍
.05; Fig. 2J). Immunostaining showed that SDF1 expression
significantly increased in BMSC and DETA-NONOate
monotherapy groups compared with the MCAo control group
(p ⬍ .05). SDF1 expression in the combination DETA-
NONOate with BMSC treatment group was significantly
increased compared with MCAo control and DETA-NONO-
ate monotherapy treatment groups, respectively (p ⬍ .05).
However, SDF1 was not significantly increased in the com-
bination DETA-NONOate with CXCR4-inhibition BMSC
treatment group compared with MCAo control (Fig. 2A–2F).
DETA-NONOate Increases BMSC CXCR4 and
MMP9 Gene and Protein Expression In Vitro
To investigate the effect of DETA-NONOate on CXCR4 and
MMP9 expression in BMSCs in vitro, CXCR4 and MMP9
gene expression and protein expression were measured in
cultured BMSCs. Figure 3 shows that DETA-NONOate treat-
ment (0.4 ␮M for 24 hours) significantly increased the num-
ber of CXCR4 (Fig. 3A, 3B, 3E) and MMP9 (Fig. 3C, 3D,
3H) immunoreactive cells compared with the nontreatment
BMSC control group (p ⬍ .05; n ⫽ 3 mice per group),
respectively. Figure 3F and 3I shows that the CXCR4 and
MMP9 gene expression in the DETA-NONOate treatment
group was significantly increased compared with the non-
treatment BMSC control group (p ⬍ .05; n ⫽ 3 mice per
group). FACS analysis shows (Fig. 3G) that the percentage of
the CXCR4 protein expression in the DETA-NONOate treat-
ment group (34.99%) was significantly increased compared
with the nontreatment BMSC control group (17.29%). SDS-
polyacrylamide gel electrophoresis zymography analysis
shows that the density of the secreted MMP9 was signifi-
cantly increased in DETA-NONOate treatment BMSCs com-
pared with nontreatment control (Fig. 3J).
Cui, Chen, Zacharek et al. 2781

Figure 2. SDF1 expression in the ischemic brain. (A–E): SDF1 immunohistochemical expression in the ischemic boundary zone at 14 days after
MCAo. (A): MCAo control; (B): BMSC treatment; (C): DETA-NONOate-alone treatment; (D): combination treatment with chemokine (CXC motif)
receptor 4-inhibition BMSCs; (E): combination treatment with normal BMSCs. (F): Quantitative SDF1 data. (G–I): Images of double immunoflu-
orescent staining of SDF1 (G) with GFAP (H). (J): SDF1 gene expression measured by real-time PCR. (K, L): Confocal images of double
immunofluorescent staining of SDF1 ([K1–K4]: 0.2 ␮m/layer; [K]: merged from [K1–K4]) with vWF ([L1–L4]: 0.2 ␮m/layer; [L]: merged from
[L1–L4]). (M): Merged image from (K, L). Scale bars ⫽ 100 ␮m (B), 50 ␮m (I). n ⫽ 9 mice per group. Abbreviations: BMSC, bone marrow stromal
cell; GFAP, glial fibrillary acidic protein; MCAo, middle cerebral artery occlusion; SDF1, stromal cell-derived factor-1; vWF, von Willebrand factor.

The SDF1/CXCR4 Axis Mediates DETA-NONOate-
Enhanced BMSC Adhesion and Migration
Delivery of BMSCs into ischemic brain is a multistep process.
The first essential step is the homing cascade, which determines
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cell adhesion to brain endothelial cell and transendothelial mi-
gration. To investigate whether DETA-NONOate increases
BMSC adhesion to brain endothelial cells or astrocytes in vitro,
measurements of BMSC adhesion to MBECs and astrocytes
2782 DETA-NONOate Enhances BMSC Migration After Stroke

Figure 3. DETA-NONOate treatment increases BMSC CXCR4 and MMP9 gene and protein expression in vitro. (A, B): CXCR4 immunostaining
in nontreatment Con (A) and DETA-NONOate treatment BMSCs (B). (C, D): MMP9 immunostaining in nontreatment Con (C) and DETA-NONOate
treatment BMSCs (D). (E, H): Quantitative data of CXCR4-positive (E) and MMP9-positive (H) cell expression. (F, I): CXCR4 (F) and MMP9 (I)
gene expression measured by real-time PCR. (G): CXCR4-positive cell expression measured by FACS. (J): MMP9 secretion measured by
zymography (n ⫽ 3 times per group). Scale bar ⫽ 50 ␮m. Abbreviations: Con, control; CXCR4, chemokine (CXC motif) receptor 4; FACS,
fluorescence-activated cell sorting; MMP9, matrix metalloproteinase 9.

were performed. Figure 4A and 4B shows that DETA-NONOate
significantly increased BMSC adhesion to MBECs and astro-
cytes compared with nontreatment control (p ⬍ .05; n ⫽ 6 wells
per group). Blocking CXCR4 with AMD3100 (20 ␮M) and
MMPs with GM6001 (10 ␮M) in BMSCs significantly attenu-
ated DETA-NONOate-induced BMSC adhesion to MBECs or
astrocytes, respectively, compared with the DETA-NONOate
treatment group (p ⬍ .05; n ⫽ 6 wells per group).
To investigate the effect of DETA-NONOate on BMSC
migration and the mechanism of DETA-NONOate regulation of
BMSC migration, a transwell culture system was used. DETA-
NONOate or SDF1␣ was added to the lower chamber. BMSCs
were placed in the upper chamber of the insert. The migration of
BMSCs into the lower chamber of the insert was measured.
Figure 4C shows that DETA-NONOate and SDF1 significantly
increased BMSC migration numbers compared with nontreat-
ment control BMSCs (p ⬍ .05; n ⫽ 4 wells per group). Blocking
CXCR4 (ADM3100) or MMP (GM6001) in BMSCs signifi-
cantly attenuated DETA-NONOate-induced BMSC migration
(p ⬍ .05; n ⫽ 4 mice per group). These data indicated that the
SDF1/CXCR4 axis, along with MMP9, regulates BMSC migra-
tion.
To further test whether DETA-NONOate promotes BMSC
migration to brain endothelial cells and astrocytes, coculture of
BMSCs with MBECs or coculture of BMSCs with astrocytes
was performed. MBECs or astrocytes were placed in the lower
chamber of the well and pretreated with or without DETA-
NONOate. BMSCs were placed in the upper chamber. BMSC
migration to the lower chamber was measured. Figure 4D shows
that DETA-NONOate significantly increased BMSC migration
toward MBECs compared with nontreatment MBECs alone
(p ⬍ .05; n ⫽ 4 wells per group). Blocking CXCR4 and MMP
in BMSCs significantly decreased DETA-NONOate-induced
BMSC migration toward MBECs (p ⬍ .05; n ⫽ 4 wells per
group). In addition, DETA-NONOate significantly increased
BMSC migration toward astrocytes compared with nontreat-
ment astrocytes alone (p ⬍ .05; n ⫽ 4 wells per group).
Blocking CXCR4 or MMP in BMSCs significantly attenuated
DETA-NONOate-induced BMSC migration toward astrocytes
(p ⬍ .05; n ⫽ 4 wells per group; Fig. 4E). These data suggest
Cui, Chen, Zacharek et al. 2783

Figure 4. Stromal cell-derived factor-1/chemokine (CXC motif) receptor 4 axis and matrix metalloproteinase mediate DETA-NONOate-induced
BMSC adhesion and migration in vitro. (A, B): BMSC adhesion assay. Coculture BMSCs with MBECs and astrocytes. BMSC adhesion to MBECs
(A) and astrocytes (B) (n ⫽ 6 wells per group). (C–E): BMSC migration assay. Culture BMSCs alone or coculture BMSCs with MBECs and
astrocytes were used. Shown are BMSC migration alone (C), BMSC migration to MBECs (D), and BMSC migration to astrocytes (E) (n ⫽ 4 wells
per group). Abbreviations: BMSC, bone marrow stromal cell; MBEC, mouse brain endothelial cell; SDF1, stromal cell-derived factor-1.

that stimulation of MBECs or astrocytes with DETA-NONOate
enhances BMSC migration. The SDF1/CXCR4 axis and MMP
regulate DETA-NONOate-induced BMSC migration.
DISCUSSION
In this study, we demonstrated that DETA-NONOate treatment
of stroke enhances BMSC migration into the ischemic brain,
which correlates with functional outcome after stroke in mice.
DETA-NONOate increases SDF1 expression in the ischemic
brain. In addition, DETA-NONOate upregulates CXCR4 and
MMP9 expression in cultured BMSCs. DETA-NONOate pro-
motes BMSC adhesion to MBECs and increases BMSC migra-
tion. The SDF1/CXCR4 axis, along with MMPs, mediates
DETA-NONOate-increased BMSC migration.
NO is a reactive molecule with numerous physiological and
pathophysiological roles affecting the nervous and cardiovascu-
lar systems. NO produced by eNOS has a crucial role in the
regulation of systemic blood pressure, vascular tone, vascular
remodeling, and angiogenesis [26]. DETA-NONOate in combi-
nation with BMSCs promotes functional outcome after stroke in
rats [13], which is consistent with our data that treatment of
stroke in adult mice with DETA-NONOate in combination with
BMSCs significantly improves functional outcome compared
with control MCAo. In this study, we demonstrate for the first
time that DETA-NONOate in combination with BMSC treat-
ment of stroke enhances BMSC migration into the ischemic
brain. In addition, the increased BMSC migration into the isch-
emic brain correlated with functional outcome after stroke.
Therefore, increased BMSC delivery to the ischemic brain im-
proves functional outcome after stroke.
www.StemCells.com
Chemokines are important factors controlling cellular mi-
gration. SDF1 has CXCR4 as its only receptor [5]. SDF1 is a
master regulator of tracking of various types of CXCR4-positive
cells [6]. Overexpression CXCR4 increases BMSC migration
toward SDF1 but does not render a survival advantage to MSCs
under serum-deprived conditions [7]. Inhibition of CXCR4 ex-
pression in BMSCs decreases BMSC migration. Thus, the in-
teraction of SDF1␣/CXCR4 likely controls the trafficking of
transplanted BMSCs. Our data show that DETA-NONOate
treatment of stroke increases endogenous brain astrocyte and
endothelial cell SDF1 expression in the ischemic border. Com-
bination treatment significantly increases SDF1 expression
compared with DETA-NONOate monotherapy and increases
the number of engrafted BMSCs compared with BMSC mono-
therapy group; however, inhibition of CXCR4 in BMSCs does
not significantly increase SDF1 expression and the number of
engrafted BMSCs. In addition, DETA-NONOate also increases
BMSC CXCR4 gene and protein expression in vitro. Inhibition
of CXCR4 expression in BMSCs significantly decreases BMSC
migration and attenuates DETA-NONOate-induced BMSC mi-
gration. Therefore, DETA-NONOate augmentation of the
SDF1/CXCR4 axis may facilitate BMSC migration into the
ischemic brain.
Circulating stem cells release factors (e.g., MMPs) that
enable them to cross the endothelial barrier and home to the
organ [6]. The CXCR4/SDF1␣ system mediates active MMP9
and MMP2 secretion from Hca-F and hepatocarcinoma cell
lines; this mediation facilitates lymphogenous metastasis [27].
SDF1 recruits osteoclast precursors partly mediated through
increases in MMP9 [28]. SDF1/CXCR4, along with MMPs,
recruits BMSCs to damaged tissues [29]. DETA-NONOate in-
creases BMSC CXCR4 and MMP9 expression. Inhibition of
CXCR4 and MMPs in BMSCs significantly decreases DETA-
NONOate-induced BMSC adhesion and migration. Therefore,
2784
DETA-NONOate induced BMSC adhesion and migration may
be mediated by the SDF1/CXCR4 and MMP pathways. We note
that the MMP inhibitor GM6001, used in this study, was not
specific for MMP9. Therefore, we cannot exclude the possibility
that MMPs other than MMP9 contribute to BMSC adhesion and
migration. In addition, MMPs may have both beneficial and
detrimental effects on developing and adult brain and spinal
cord. Detrimental effects include dysfunction of the blood-brain
barrier, demyelination, neuroinflammation, and neurotoxicity
[30, 31]. Beneficial effects include roles in the development and
neurogenesis of the central nervous system, growth and regen-
eration of axons, myelogenesis, angiogenesis, and termination
of neuroinflammation [21, 32, 33]. The double-edged effects of
MMP9 depend on the temporal and spatial distribution of
MMP9 within the cells of the neurovascular unit [31]. MMP9
contributes to damage early after a stroke [34]. In addition,
MMP9 takes on a reparative role days after stroke [32].
On the basis of our data, the following additional studies are
warranted. Testing of a ceiling effect to identify the limit of
therapeutic efficacy of using higher doses of BMSCs and
DETA-NONOate would be informative and potentially clini-
cally relevant. In addition, our studies in no way exclude the
possibility that other chemokines and growth factors (e.g., he-
patocyte growth factor, platelet-derived growth factor, and an-
giogenic factors [e.g., vascular endothelial growth factor
(VEGF)/VEGF receptor 2 and Angiopoietin 1/Tie2]) also con-
tribute to BMSC migration into the ischemic brain and are likely
increased by DETA-NONOate [35, 36]. Upregulation of circu-
REFERENCES
1 Dezawa M, Hoshino M, Nabeshima Y et al. Marrow stromal cells:
Implications in health and disease in the nervous system. Curr Mol Med
2005;5:723–732.
2 Li Y, Chen J, Chen XG et al. Human marrow stromal cell therapy for
stroke in rat: Neurotrophins and functional recovery. Neurology 2002;
59:514 –523.
3 Chen J, Li Y, Wang L et al. Therapeutic benefit of intravenous admin-
istration of bone marrow stromal cells after cerebral ischemia in rats.
Stroke 2001;32:1005–1011.
4 Muller-Ehmsen J, Krausgrill B, Burst V et al. Effective engraftment but
poor mid-term persistence of mononuclear and mesenchymal bone mar-
row cells in acute and chronic rat myocardial infarction. J Mol Cell
Cardiol 2006;41:876 – 884.
5 Bagri A, Gurney T, He X et al. The chemokine SDF1 regulates migration
of dentate granule cells. Development 2002;129:4249 – 4260.
6 Kucia M, Reca R, Miekus K et al. Trafficking of normal stem cells and
metastasis of cancer stem cells involve similar mechanisms: Pivotal role
of the SDF-1-CXCR4 axis. STEM CELLS 2005;23:879 – 894.
7 Bhakta S, Hong P, Koc O. The surface adhesion molecule CXCR4
stimulates mesenchymal stem cell migration to stromal cell-derived
factor-1 in vitro but does not decrease apoptosis under serum deprivation.
Cardiovasc Revasc Med 2006;7:19 –24.
8 Ji JF, He BP, Dheen ST et al. Interactions of chemokines and chemokine
receptors mediate the migration of mesenchymal stem cells to the im-
paired site in the brain after hypoglossal nerve injury. STEM CELLS
2004;22:415– 427.
9 Cherla RP, Ganju RK. Stromal cell-derived factor 1 alpha-induced che-
motaxis in T cells is mediated by nitric oxide signaling pathways.
J Immunol 2001;166:3067–3074.
10 Aicher A, Heeschen C, Mildner-Rihm C et al. Essential role of endo-
thelial nitric oxide synthase for mobilization of stem and progenitor cells.
Nat Med 2003;9:1370 –1376.
11 Janowska-Wieczorek A, Marquez LA, Dobrowsky A et al. Differen-
tial MMP and TIMP production by human marrow and peripheral
blood CD34(⫹) cells in response to chemokines. Exp Hematol 2000;
28:1274 –1285.
12 Zhang R, Wang L, Zhang L et al. Nitric oxide enhances angiogenesis via
the synthesis of vascular endothelial growth factor and cGMP after stroke
in the rat. Circ Res 2003;92:308 –313.
13 Chen J, Li Y, Zhang R et al. Combination therapy of stroke in rats with
a nitric oxide donor and human bone marrow stromal cells enhances
angiogenesis and neurogenesis. Brain Res 2004;1005:21–28.
DETA-NONOate Enhances BMSC Migration After Stroke
lating progenitor endothelial cells by exogenous cell and phar-
macological monotherapies may also enhance functional recov-
ery from stroke [2, 3, 13, 37– 41]. Whether these endogenous
progenitor cells are enhanced by combination treatment is un-
known.
SUMMARY
DETA-NONOate treatment of stroke enhances BMSC migra-
tion into the ischemic brain by increasing SDF1/CXCR4 and
MMP. Priming of the ischemic brain with DETA-NONOate
enhances the efficacy of BMSC therapy.
ACKNOWLEDGMENTS
We thank Qinge Lu for technical assistance. This work was
supported by National Institute of Neurological Disorders and
Stroke (NINDS) Grant RO1-NS047682, American Heart Asso-
ciation Grant 0750048Z, and NINDS Grant PO1-NS23393.
DISCLOSURE OF POTENTIAL CONFLICTS
OF INTEREST
The authors indicate no potential conflicts of interest.
14 Chen J, Zhang C, Jiang H et al. Atorvastatin induction of VEGF and
BDNF promotes brain plasticity after stroke in mice. J Cereb Blood Flow
Metab 2005;25:281–290.
15 Watanabe M, Matsuyama W, Shirahama Y et al. Dual effect of
AMD3100, a CXCR4 antagonist, on bleomycin-induced lung inflamma-
tion. J Immunol 2007;178:5888 –5898.
16 Li Y, Jiang N, Powers C et al. Neuronal damage and plasticity identified
by microtubule-associated protein 2, growth-associated protein 43, and
cyclin D1 immunoreactivity after focal cerebral ischemia in rats. Stroke
1998;29:1972–1980; discussion 1980 –1981.
17 Chen J, Li Y, Katakowski M et al. Intravenous bone marrow stromal cell
therapy reduces apoptosis and promotes endogenous cell proliferation
after stroke in female rat. J Neurosci Res 2003;73:778 –786.
18 Shen LH, Li Y, Chen J et al. Therapeutic benefit of bone marrow stromal
cells administered 1 month after stroke. J Cereb Blood Flow Metab
2007;27:6 –13.
19 Livak KJ, Schmittgen TD. Analysis of relative gene expression data
using real-time quantitative PCR and the 2(-delta delta C(T)) method.
Methods 2001;25:402– 408.
20 Romanic AM, White RF, Arleth AJ et al. Matrix metalloproteinase
expression increases after cerebral focal ischemia in rats: Inhibition of
matrix metalloproteinase-9 reduces infarct size. Stroke 1998;29:
1020 –1030.
21 Wang L, Zhang ZG, Zhang RL et al. Matrix metalloproteinase 2 (MMP2)
and MMP9 secreted by erythropoietin-activated endothelial cells pro-
mote neural progenitor cell migration. J Neurosci 2006;26:5996 – 6003.
22 Fernandez-Patron C, Zouki C, Whittal R et al. Matrix metalloproteinases
regulate neutrophil-endothelial cell adhesion through generation of en-
dothelin-1 1 . FASEB J 2001;15:2230 –2240.
32
23 Wang L, Li Y, Chen X et al. MCP-1, MIP-1, IL-8 and ischemic cerebral
tissue enhance human bone marrow stromal cell migration in interface
culture. Hematology 2002;7:113–117.
24 Annabi B, Thibeault S, Lee YT et al. Matrix metalloproteinase regulation
of sphingosine-1-phosphate-induced angiogenic properties of bone mar-
row stromal cells. Exp Hematol 2003;31:640 – 649.
25 Zhou B, Han ZC, Poon MC et al. Mesenchymal stem/stromal cells
(MSC) transfected with stromal derived factor 1 (SDF-1) for therapeutic
neovascularization: enhancement of cell recruitment and entrapment.
Med Hypotheses 2007;68:1268 –1271.
26 Chen J, Zacharek A, Zhang C et al. Endothelial nitric oxide synthase
regulates brain-derived neurotrophic factor expression and neurogenesis
after stroke in mice. J Neurosci 2005;25:2366 –2375.
27 Chu H, Zhou H, Liu Y et al. Functional expression of CXC chemokine
recepter-4 mediates the secretion of matrix metalloproteinases from
Cui, Chen, Zacharek et al.
mouse hepatocarcinoma cell lines with different lymphatic metastasis
ability. Int J Biochem Cell Biol 2007;39:197–205.
28 Yu X, Huang Y, Collin-Osdoby P et al. Stromal cell-derived factor-1
(SDF-1) recruits osteoclast precursors by inducing chemotaxis, matrix
metalloproteinase-9 (MMP-9) activity, and collagen transmigration.
J Bone Miner Res 2003;18:1404 –1418.
29 Son BR, Marquez-Curtis LA, Kucia M et al. Migration of bone marrow
and cord blood mesenchymal stem cells in vitro is regulated by stromal-
derived factor-1-CXCR4 and hepatocyte growth factor-c-met axes and
involves matrix metalloproteinases. STEM CELLS 2006;24:1254 –1264.
30 Asahi M, Wang X, Mori T et al. Effects of matrix metalloproteinase-9
gene knock-out on the proteolysis of blood-brain barrier and white matter
components after cerebral ischemia. J Neurosci 2001;21:7724 –7732.
31 Zlokovic BV. Remodeling after stroke. Nat Med 2006;12:390 –391.
32 Zhao BQ, Wang S, Kim HY et al. Role of matrix metalloproteinases in
delayed cortical responses after stroke. Nat Med 2006;12:441– 445.
33 Sun CY, Hu Y, Wang HF et al. Brain-derived neurotrophic factor
inducing angiogenesis through modulation of matrix-degrading pro-
teases. Chin Med J 2006;119:589 –595.
34 Kim YS, Lee KY, Koh SH et al. The role of matrix metalloproteinase 9
in early neurological worsening of acute lacunar infarction. Eur Neurol
2006;55:11–15.
www.StemCells.com
2785
35 Zacharek A, Chen J, Zhang C et al. Nitric oxide regulates Angiopoietin1/
Tie2 expression after stroke. Neurosci Lett 2006;404:28 –32.
36 Zacharek A, Chen J, Cui X et al. Angiopoietin1/Tie2 and VEGF/Flk1
induced by MSC treatment amplifies angiogenesis and vascular stabili-
zation after stroke. J Cereb Blood Flow Metab 2007 [Epub ahead of
print].
37 Zhang ZG, Zhang L, Jiang Q et al. VEGF enhances angiogenesis and
promotes blood-brain barrier leakage in the ischemic brain. J Clin Invest
2000;106:829 – 838.
38 Zhang ZG, Zhang L, Tsang W et al. Correlation of VEGF and angio-
poietin expression with disruption of blood-brain barrier and angiogen-
esis after focal cerebral ischemia. J Cereb Blood Flow Metab 2002;22:
379 –392.
39 Chen J, Zhang ZG, Li Y et al. Intravenous administration of human bone
marrow stromal cells induces angiogenesis in the ischemic boundary
zone after stroke in rats. Circ Res 2003;92:692– 699.
40 Zhang Z, Chopp M. Vascular endothelial growth factor and angiopoietins
in focal cerebral ischemia. Trends Cardiovasc Med 2002;12:62– 66.
41 Chen J, Zacharek A, Li A et al. Vascular endothelial growth factor
mediates atorvastatin-induced mammalian achaete-scute homologue-1
gene expression and neuronal differentiation after stroke in retired
breeder rats. Neuroscience 2006;141:737–744.