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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

Departments of ^aNeurology and
^bBiostatistics and Research Epidemiology, Henry Ford Health Sciences Center, Detroit, Michigan, USA;
^cDepartment of Neurobiology, Theradigm, Inc., Baltimore, Maryland, USA;
^dDepartment 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

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{at}neuro.hfh.edu

Received on March 9, 2007; accepted for publication on July 12, 2007.

First published online in STEM CELLS EXPRESS  July 19, 2007.

	ABSTRACT

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
Stromal cell-derived factor-1 (SDF1) and its chemokine (CXC motif) receptor 4 (CXCR4), along with matrix metalloproteinases (MMPs), regulate bone marrow stromal cell (BMSC) migration. We tested the hypothesis that a nitric oxide donor, DETA-NONOate, increases endogenous ischemic brain SDF1 and BMSC CXCR4 and MMP9 expression, 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 = 9 mice per group): (a) phosphate-buffered saline; (b) BMSCs (5 x 10⁵); (c) 0.4 mg/kg DETA-NONOate; (d) combination of CXCR4-inhibition BMSCs with DETA-NONOate; and (e) combination of BMSCs with DETA-NONOate. To elucidate the mechanisms underlying combination-enhanced BMSC migration, transwell cocultures of BMSC with mouse brain endothelial cells (MBECs) or astrocytes were performed. Combination treatment significantly improved functional outcome 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 significantly increased BMSC CXCR4 and MMP9 expression and promoted BMSC adhesion and migration to MBECs and astrocytes compared with nontreatment BMSCs. Inhibition 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.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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The regenerative potential of bone marrow stromal cells (BMSCs) has been demonstrated in myocardial, limb, and brain ischemia [1]. Systemically administered BMSCs selectively migrate 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 engrafted 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 treatment of stroke.

Chemokines are important factors controlling cellular migration. Stromal cell-derived factor-1 (SDF1) and its unique receptor [5] chemokine (CXC motif) receptor 4 (CXCR4) regulate 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]. Intracerebral 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 trafficking of transplanted BMSCs. In addition, matrix metalloproteinases (MMPs) are involved in SDF1/CXCR4-induced chemotaxis of human hematopoietic progenitor cells across subendothelial basement membranes [11]. SDF1 gradient increases the chemotaxis of bone marrow and blood CD34(+) cells, which is blocked by inhibitors of MMPs [11].

DETA-NONOate ([Z]-1-[N-(2-aminoethyl)-N-(2-ammonioethyl) 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-NONOate amplified restorative therapy after stroke in mice. We hypothesized that DETA-NONOate treatment promotes BMSC migration into the ischemic brain by increasing SDF1 within the compromised brain.

	MATERIALS AND METHODS

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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-2'-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 µg/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 transient (2.5 hours) monofilament right middle cerebral artery occlusion (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 x 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 x 10⁵) pretreated with specific CXCR4 inhibitor AMD3100 [15] (20 µM for 3 hours) with DETA-NONOate (0.4 mg/kg); or (e) combination of BMSCs (5 x 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 x 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 (single injection) and 5 x 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 paraffin [14]. A standard paraffin block was obtained from the center of the lesion (bregma, –1 mm to +1 mm). A series of 6-µm-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, respectively. 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 microscopy (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 contralateral hemispheres were counted. For quantification, the percentage 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 µM 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 (Applied Biosystems, Foster City, CA, http://www.appliedbiosystems.com) using three-stage program parameters provided by the manufacturer. 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; reverse, AGC TGA TTG ACT AAA GTA GCT GGA).

SDS-Polyacrylamide Gel Electrophoresis Zymography
Conditioned media were collected and concentrated using a Centricon concentrator (Millipore, Bedford, MA, http://www.millipore.com). Protein concentrations were analyzed with a Bio-Rad system. Equal amounts of protein (20 µg/lane) for each sample were mixed with 2x sample buffer and loaded on a 10% polyacrylamide gel incorporated with 0.1% gelatin for electrophoresis. MMP9 zymographic standards were used as positive controls (Chemicon, Temecula, 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 visualized as a transparent band against a blue background. Zymography was measured for quantification analysis by spot density measurement 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 x 10⁶) were incubated, fixed with 4% paraformaldehyde, and rinsed twice with 1x PBS with CXCR4 antibody (1:250; AB1846; Chemicon) conjugated with FITC (1:200; Jackson Immunoresearch Laboratories). Immunostaining analysis 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 signaling pathway of DETA-NONOate upregulation of BMSC adhesion, green fluorescent protein (GFP)-BMSCs were cultured with mouse brain endothelial cells (MBECs) (American Type Culture Collection, Manassas, VA, http://www.atcc.org) and astrocytes (American Type Culture Collection). MBECs or astrocytes (1 x 10⁴ cells per well) were placed in 96-well chambers and pretreated with or without DETA-NONOate (0.4 µM) for 24 hours, separately. Then, BMSCs were added to MBECs or astrocytes and treated with or without AMD3100 (20 µM; AnorMED, Groton, CT, http://www.anormed.com), a specific antagonist of CXCR4, or GM6001 (10 µM; 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 µM) for 24 hours. The number of BMSCs migrating to the lower chamber was counted.

To further test whether DETA-NONOate promotes BMSC migration toward MBECs or astrocytes, transwell coculture of GFP-BMSCs with MBECs or GFP-BMSCs with astrocytes was performed. MBECs or astrocytes (5 x 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 µM) for 24 hours, separately. In addition, 24-well Transwell polycarbonate inserts with a pore size of 8 µm (Corning Life Sciences) coated with 50 µg/ml fibronectin (Chemicon) and 0.1% gelatin were used. GFP-BMSCs (5 x 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 x 10⁴ cells per well) were placed in the lower chamber and pretreated with or without DETA-NONOate (0.4 µM) for 24 hours. GFP-BMSCs were pretreated with or without AMD3100 (20 µM) or GM6001 (10 µM) 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 comparisons were performed if the overall treatment effect was significant 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

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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 combination treatment of stroke improves neurological functional recovery, a battery of functional tests were performed. Figure 1A and 1B shows that combination DETA-NONOate with BMSCs significantly 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 combination with DETA-NONOate treatment group compared with MCAo control.

View larger version (55K): [in this window] [in a new window]   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 µm. 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.

Combination Treatment with DETA-NONOate and BMSCs Promotes SDF1 Expression in the Ischemic Boundary Zone
The SDF1/CXCR4 axis regulates BMSC recruitment to lesioned area [25]. To test whether DETA-NONOate enhancement 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 compared 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-NONOate monotherapy treatment groups, respectively (p < .05). However, SDF1 was not significantly increased in the combination DETA-NONOate with CXCR4-inhibition BMSC treatment group compared with MCAo control (Fig. 2A–2F). Double immunofluorescent staining showed that SDF1 primarily 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).

View larger version (84K): [in this window] [in a new window]   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 immunofluorescent 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 µm/layer; [K]: merged from [K1–K4]) with vWF ([L1–L4]: 0.2 µm/layer; [L]: merged from [L1–L4]). (M): Merged image from (K, L). Scale bars = 100 µm (B), 50 µm (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.

View larger version (41K): [in this window] [in a new window]   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 µm. Abbreviations: Con, control; CXCR4, chemokine (CXC motif) receptor 4; FACS, fluorescence-activated cell sorting; MMP9, matrix metalloproteinase 9.

View larger version (32K): [in this window] [in a new window]   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.

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 nontreatment 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 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

Top Abstract Introduction Materials and Methods Results Discussion Summary Disclosure of Potential... Acknowledgments References

 
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 promotes BMSC adhesion to MBECs and increases BMSC migration. 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 cardiovascular 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 combination 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 treatment of stroke enhances BMSC migration into the ischemic brain. In addition, the increased BMSC migration into the ischemic brain correlated with functional outcome after stroke. Therefore, increased BMSC delivery to the ischemic brain improves functional outcome after stroke.

Chemokines are important factors controlling cellular migration. 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 expression in BMSCs decreases BMSC migration. Thus, the interaction 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. Combination treatment significantly increases SDF1 expression compared with DETA-NONOate monotherapy and increases the number of engrafted BMSCs compared with BMSC monotherapy 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 migration. 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 increases BMSC CXCR4 and MMP9 expression. Inhibition of CXCR4 and MMPs in BMSCs significantly decreases DETA-NONOate-induced BMSC adhesion and migration. Therefore, 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 regeneration 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 clinically relevant. In addition, our studies in no way exclude the possibility that other chemokines and growth factors (e.g., hepatocyte growth factor, platelet-derived growth factor, and angiogenic factors [e.g., vascular endothelial growth factor (VEGF)/VEGF receptor 2 and Angiopoietin 1/Tie2]) also contribute to BMSC migration into the ischemic brain and are likely increased by DETA-NONOate [35, 36]. Upregulation of circulating progenitor endothelial cells by exogenous cell and pharmacological monotherapies may also enhance functional recovery from stroke [2, 3, 13, 37–41]. Whether these endogenous progenitor cells are enhanced by combination treatment is unknown.

	SUMMARY

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DETA-NONOate treatment of stroke enhances BMSC migration into the ischemic brain by increasing SDF1/CXCR4 and MMP. Priming of the ischemic brain with DETA-NONOate enhances the efficacy of BMSC therapy.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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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 Association Grant 0750048Z, and NINDS Grant PO1-NS23393.

	REFERENCES

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