Deferoxamine preconditioning potentiates mesenchymal stem cell homing in vitro ...

1. Introduction

2. Methods

3. Results

4. Discussion

Original Research

Deferoxamine preconditioningpotentiates mesenchymal stemcell homing in vitro and instreptozotocin-diabetic ratsR Najafi & Ali M Sharifi††Tehran University of Medical Sciences, School of Medicine, Razi Drug Research Center,

Department of Pharmacology, Tehran, Iran

Objective: Today, cell therapy is considered a promising alternative in treat-

ment of several diseases such as type 1 diabetes. Loss of transplanted stem

cell and more importantly scarcity in the number of cells reaching to target

tissue is a major obstacle in cell therapy. There is evidences showing that

deferoxamine (DFO), an iron chelator, increases the mobilization and homing

of progenitor cells through increasing the stability of hypoxia-inducible factor

1a (HIF-1a) protein. In this study, the effect of DFO on some factors involved

in homing of bone marrow-derived mesenchymal stem cell was investigated,

and the other objectives of this research were to determine whether DFO is

able to increase migration and subsequent homing of mesenchymal stem

cell (MSCs) both in vitro and in vivo in streptozotocin-diabetic rats.

Research design and methods:MSCs were treated by DFO in minimal essential

medium a (aMEM) for 24 h. The expression and localization of HIF-1a were

evaluated by western blotting and immunocytochemistry. The expression of

C-X-C chemokine receptor type 4 (CXCR-4) and chemokine receptor 2 (CCR2)

were assessed by western blotting and RT-PCR. The activity of matrix metallo-

proteinases (MMP) -2 and -9 were measured by gelatin zymography. Finally,

in vitro migration of MSCs toward different concentrations of stromal

cell-derived factor and monocyte chemotactic protein-1 were also evaluated.

To demonstrate the homing of MSCs in vivo, DFO-treated chloromethyl-

benzamidodialkylcarbocyanine-labeled MSCs were injected into the tail vein

of rats, and the number of stained MSCs reaching to the pancreas were

determined after 24 h.

Results: In DFO-treated MSCs, expression of HIF-1a (p < 0.001), CXCR4

(p < 0.001), CCR2 (p < 0.001), and the activity of MMP-2 (p < 0.01) and

MMP-9 (p < 0.05) were significantly increased compared to control groups.

Elevation of HIF-1a, upregulation of CXCR4/CCR2 and higher activity of

MMP-2/MMP-9 in DFO-treated MSCs were reversed by 2-methoxyestradiol

(2-ME; 5 µmol), a HIF-1a inhibitor. The in vitro migrations as well as in vivo

homing of DFO-treated MSCs were also significantly higher than control

groups (p < 0.05).

Conclusions: Preconditioning of MSCs by DFO prior to transplantation could

increase homing of MSCs through affecting some chemokine receptors as

well as proteases involved and eventually improving the efficacy of

cell therapy.

Keywords: cell therapy, deferoxamine, hypoxia-inducible factor 1a, mesenchymal stem cell

homing, type 1 diabetes

Expert Opin. Biol. Ther. (2013) 13(7):959-972

10.1517/14712598.2013.782390 © 2013 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682 959All rights reserved: reproduction in whole or in part not permitted

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1. Introduction

Destruction of insulin-producing b-cells in the pancreas is themain feature of type 1 diabetes, which results in increased levelsof blood glucose [1,2]. Presently, the most common treatmentused for type 1 diabetic patients is insulin therapy, but in mostcases this type of therapy is unable to regulate and control bloodglucose [3]. The alternative method to treat diabetes are trans-plantation of whole pancreas and/or pancreatic islet cells [4].However, inadequate organ donor and adverse effects of immu-nosuppressive drugs are unsolved consequences of pancreatictransplantation [5]. The main challenge at present is the estab-lishment of procedures to provide the safe and effective methodfor the prevention as well as treatment of type 1 diabetes [6].Currently, among others, adult stem cell therapy particu-

larly using bone marrow-derived mesenchymal stem cells(BM-MSCs) has been one of the best proposed options forthe treatment of a variety of diseases [7,8]. Since mesenchymalstem cells (MSCs) could easily be isolated from bone marrowand differentiated into a variety of cell types, being known asthe most commonly used stem cells in tissue engineering andregenerative medicine [9,10]. Regeneration capability as well asimmunomodulating property of MSCs makes them suitabletools for cellular medicine [11]. However, an efficient cell ther-apy requires transmigration of MSCs across the endotheliumand reaching the injured tissue by a process called homing.Reports from many studies indicate that during stem cell ther-apy, only a small percentage of transplanted cells reach the tar-get tissue [7,12]. Homing process has several steps, including:chemokine-chemokine receptor interactions, attachment toendothelial cells and migration across the endothelium [13].C-X-C chemokine receptor type 4 (CXCR4) chemokine cellsurface receptor and its ligand stromal cell-derived factor 1(SDF-1) play an important role in migration and homingof MSCs to the damage tissue and are expressed inMSCs [14]. Other chemokine receptor that is also expressedby MSCs is chemokine receptor 2 (CCR2), which is a majorreceptor for monocyte chemotactic protein-1 (MCP-1) thatmodulates monocytes and macrophage recruitment duringinflammatory responses [15].Moreover, other important factorssecreted by MSCs are proteases such as matrix metalloprotei-nases (MMPs) functioning by degrading the extracellularmatrix (ECM) during chemotaxis [16].Deferoxamine (DFO) is an iron-chelating drug widely used

in the treatment of chronic iron overload [17,18]. Prolyl-4 hydroxylase is the enzyme inhibited by DFO. The onlyfunction for the prolyl-4 hydroxylases is hydroxylation of pro-line residue, leading to hypoxia-inducible factor 1a (HIF-1a)ubiquitination and proteasomal degradation. For activation,this enzyme needs oxygen, iron and 2-oxoglutarate as cofac-tors, among which, iron and 2-oxoglutarate could be impor-tant drug targets for inhibition of the prolyl hydroxylases(PHDs) under normoxic conditions [19,20].HIF-1a is a transcription factor and key regulator of the

cellular response to the changes in oxygen concentration [21].

Under hypoxic conditions, HIF-1a, through binding to hor-mone response elements, increases transcription of genes thatmay act as a chemoattractant for stem cells and stimulateregeneration including vascular endothelial growth factor(VEGF), erythropoietin, SDF-1 and CXCR4 [19,22]. Accord-ingly, stabilization of HIF-1a is essential for the proper func-tioning of this transcription factor as a regulator of geneexpression under hypoxia [6,23]. Therefore, DFO has beenextensively used as a hypoxia mimetic agent in normoxia, sta-bilizing HIF-1a. A previous study has showed that pretreat-ment of human neural stem cells (NSCs) with DFO canprotect the rodent brain from ischemia and improves the func-tion of NSCs by induction of some chemokine receptorsinvolved in the migration and homing process such asSDF-1a/CXCR4 [19]. Other evidence show that DFO alsostimulates the migration and enhances homing of endothelialprogenitor cells (EPCs) to ischemic vasculature by stabilizationof HIF-1a [24].

Moreover, studies in human cardiac fibroblasts, neonatalrat ventricular myocytes and human umbilical vein endothe-lial cells, have shown that DFO can increase VEGF mRNAexpression. VEGF has also been shown to play an importantrole in the homing of EPCs [25]. On the basis of these obser-vations, we decided to evaluate the preconditioning effect ofDFO on HIF-1a and also some essential factors that involvein migration as well as homing of MSCs both in vitro andin vivo in streptozotocin (STZ)-diabetic rats, in order toenhance the efficacy of cell therapy in type 1 diabetes.

2. Methods

2.1 Antibodies and chemicalsPolyclonal antibodies to CXCR4 and CCR2 were purchasedfrom Abcam (Cambridge, UK). Antibodies against fluores-cein isothiocyanate (FITC)-conjugated CD31, CD44, CD45and CD90were fromBDBiosciences Pharmingen (SanDiego,CA, USA) and antibody against FITC-conjugated CD34,CD105 and IgG1 were from Santa Cruz Biotechnology(CA, USA). Antibody against FITC-conjugated CD106 andCD11b were purchased from Biolegend (San Diego, CA,USA). Monoclonal antibody anti-HIF-1a, Hoechst, DAPI,STZ, gelatin A and gelatin B, protease and phosphatase inhib-itor cocktails, DFO, 2-methoxyestradiol (2-ME) and MTTwere purchased from Sigma (Sigma Aldrich, St Louis, MO,USA). The minimal essential medium a (aMEM), fetalbovine serum (FBS), penicillin--streptomycin were purchasedfrom Gibco (Carlsbad, CA, USA). Trizol reagent was fromInvitrogen (Merelbeke, Belgium) and Oligo (dT) primer andmolony murine leukemia virus reverse transcriptase (MMLV)was purchased from Fermentas (UK). Horseradish peroxidaselinked anti-rabbit secondary antibody and anti-b-actin wereobtained from Cell Signaling (Danvers, MA, USA). Recombi-nant rat SDF-1 and MCP-1 were from Biomol (Enzo Life Sci-ences, Inc., USA). Chemicon QCM 96-well cell migrationassay was purchased from Millipore (Billerica, MA, USA).

R. Najafi & A. M. Sharifi

960 Expert Opin. Biol. Ther. (2013) 13(7)

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The enhanced chemiluminescence (ECL) detection kit wasfrom Amersham Biosciences (Amersham, Buckinghamshire,UK) and CM-Dil was obtained from Invitrogen (USA).

2.2 Experimental animalsMale Wistar strain rats with 8 -- 12 weeks of age and weighing180 -- 250 g were obtained from the animal facility of the Pas-ture Institute of Iran. Rats were housed in individual standardcages at a temperature of 20 -- 22�C and 45 -- 55% humidityunder a 12/12 h light/dark cycle. All rats were fed withstandard rodent diet and water. For surgery, animals wereanesthetized with intraperitoneal injections of 50 mg/kgketamine and 5 mg/kg xylazine. All attempts were made tominimize animal suffering and to reduce the number of ani-mals used. This study was approved by Ethical Committeeof Tehran University of Medical Sciences base on NationalInstitutes of Health Principles of Laboratory Animal Care(NIH publication no. 85-23, revised 1985).

2.3 Rat MSCs isolation, culture and treatment

with DFORats were anesthetized by ketamin-xylazin and then placed inethanol (70% [v/v]). After dissecting out the femur and tibia,bone marrow was aspirated with 22 gauge needle, and cellsresuspended in aMEM supplemented with 15% FBS and1% penicillin--streptomycin then filtered through a 70 µmmesh. Cells were centrifuged at 1,200 rpm for 6 min, andcell pellets were cultured in a 25-cm2 flask in 5 ml aMEMmedium containing 15% FBS and antibiotics. The cellswere incubated in a humidified 5% CO2 incubator at 37

�C.Non-adherent cells were removed after 48 h and culturemedium was changed every 2 -- 3 days.All experiments wereperformed on cells from passaged 2 -- 4. DFO was dissolvedin distilled water, which was then diluted in culture medium(aMEM) immediately before use. MSCs were cultured inaMEM at 37�C in 5% CO2 for 24 h in serum-free media con-taining DFO. Cultures without DFO served as controls. Toconfirm the role of HIF-1a-mediated regulation of CXCR4and CCR2 expression and MMP-2/MMP-9 activity, cellswere pretreated with 2-ME2, a HIF-1a inhibitor, for 24 h.

2.4 Flow cytometry analysis of MSCsCultured MSCs were detached by trypsin/EDTA and washedtwice with PBS. About 2 � 105 cells were incubated with anappropriate concentration of FITC-conjugated monoclonalrat antimouse CD11b, CD31, CD34, CD44, CD45,CD73, CD90, CD105 and CD106 for 40 min at 4�C inthe dark. Cells were washed by centrifugation for 5 minsand resuspended in PBS. Quantitative fluorescence analysiswas carried out using FACS caliber cytometer (Becton Dick-inson, San Diego, CA, USA) and CellQuest software. At least20,000 events were collected. All experiments also incubatedwith FITC-rat anti-mouse IgG1 as a negative isotype control.

2.5 Cell viability assayCell viability was determined using the MTT colorimetricassay. This colorimetric assay is based on the cleavage oftetrazolium salts to form insoluble purple formazan in meta-bolically active cells that can be quantified using a spectropho-tometer. The MSCs were plated at the density of 5,000 perwell in a 96-micro plate well, and then cells were treatedwith various concentrations of DFO (0, 75, 150, 300 µg/ml).Serum-free media served as a negative control and freshaMEM supplemented with 15% FBS served as a positive con-trol. After 24, 48 and 72 h incubation, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (0.5 mg/ml) was addedinto each well and were incubated for 2 h at 37�C. Cell culturemedium was removed, and then 100 µl dimethyl sulfoxide wasadded. The rate of absorption was measured at 570 nm, with630 nm as a reference in an ELISA reader.

2.6 RT-PCRTotal RNA was isolated from cultured MSCs using TRIZOLreagent according to the manufacturer’s instructions and wasspectrophotometrically quantified. First-strand cDNA was syn-thesized with 1 µg RNA in the presence of 2 µM oligo dTprimer and 200 U MMLV in a total volume of 20 µL. Thereaction mixture was incubated for 1 h at 42�C then followedby incubation at 72�C for 10 min. Aliquots of 5 µL ofcDNA were subjected to PCR utilizing specific primers:CXCR4 sense primer, 5¢-GGAAGGAACTGAACGCTCCAGAA-3¢ and antisense primer, 5¢-GAAACCACACAGCACAACCAAAC-3¢; CCR2 sense primer, 5¢-TGATCCTGCCCCTACTTGTCAT-3¢ and anti-sense primer, 5¢-ATGGCCTGGTCTAAGTGCATGT-3¢ and b-actin sense primer, 5¢-TGTCCACCTTCCAGCAGATGT-3¢ and anti-sense primer, 5¢-AGCTCAGTAACAGTCCGCCTAGA-3¢. The PCR conditionsfor both primer sets were as follows: hot start at 94�C for5 min; 30 amplification cycles, each consisting of 94�C for30 s, 55�C for 1 min and 72�C for 42 s, and a final extensionstep at 72�C for 5 min for CCR2. PCR for CXCR4 was per-formed at 94�C, 52�C and 72�C. PCR products were sepa-rated on 1.5% agarose gels and visualized by Nancy staining.

2.7 Western blot analysisCultures of confluent MSCs were exposed to DFO (150 µM)for 0, 8, 16 and 24 h. The untreated and treated cultured cellswere trypsinized, washed twice with ice-cold PBS and lysed in0.2 ml of RIPA buffer (10 mM Tris--HCl, pH 7.4, 150 mMNaCl, 5 mM EDTA, 1% Triton X-100, 0.1% sodiumdodecyl sulfate and 0.5% sodium deoxycholate) containingprotease and phosphatase inhibitor cocktails and centrifugedat 15,000 g for 20 min at 4�C. The protein concentration wasdetermined using the Bradford method [26]. For immunoblot-ting, equal amounts of proteins, 50 µg from each sample,were loaded and separated by SDS-PAGE [27] and transferredonto a polyvinylidene difluoride (PVDF) membrane. Themembrane then was incubated with the following primaryantibodies: mouse monoclonal anti-HIF-1a (1:1,000), Rabbit

DFO preconditioning potentiates MSC homing in vitro and in STZ-diabetic rats

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polyclonal anti-CXCR4 (1:1,000) and rabbit polyclonal anti-CCR2 (1:1,000). Then the membranes were incubated for 1 hat room temperature with the secondary antibody (anti-mouseIgG and anti-rabbit antibody conjugatedwith horse-radish perox-idase). To control for protein loading, the membranes wereprobed with a rabbit anti-b-actin antibody. The protein bandswere visualized using ECL detection reagents and quantified bydensitometric analysis (TotalLab software, Wales, UK).

2.8 Gelatin zymographyMMP-9 and MMP-2 activity was assessed by zymography.MSCs were grown in serum-free medium. Supernatantswere collected and mixed 1:1 with sample buffer (250 mMTris-HCl, 40% v/v glycerol, 8% w/v SDS and 0.01% w/vbromophenol blue, pH 6.8). The samples were then run at10% polyacrylamide gel copolymerized with 2 mg/ml gelatinA and gelatin B in the presence of 0.1% SDS under non-reducing conditions at a constant voltage of 125 V for1 -- 2 h. The gel was then placed in 2.5% Triton X-100 toremove SDS. Subsequently, the gel was placed on developingbuffer (50 mM Tris--HCl, 200 mM NaCl, 5 mM CaCl2 and0.01% NaN3, pH 7.5) for 24 h at 37�C to optimize metallo-proteinase activity. The gel was stained for 1 h in coomassieblue solution containing 40% methanol, 10% acetic acid,0.1% coomassie brilliant blue G-250 and deionized water.The gels were destained in 40% methanol, 10% acetic acidand deionized water. A clear band against a blue backgroundrepresents enzyme activity. Molecular weights of the protei-nases were determined by comparison to protein molecularweight standards (Bio-Rad). Finally, gels were photographedand analyzed by NIH Image J software.

2.9 ImmunocytochemistryCultured cells were washed twice with PBS and the cells werefixed in 4% paraformaldehyde in PBS for 20 min. The cellswere permeabilized with PBS containing 0.25% TritonX-100 for 20 min. The cells were incubated in the mousemonoclonal anti-HIF-1a (1:500) antibody in 1% BSA inPBS for 24 h at 4�C. The slides were rinsed with PBS andthen incubated for 1 h with FITC-conjugated goat anti-mouse in 1% BSA for 1 h at room temperature in dark. Wedecanted the secondary antibody and washed it three timeswith PBS for 5 min then incubated the cells for 1 -- 5 minin the diluted DAPI (0.1 -- 1 µg/ml). The cells were thenwashed three times with PBS and examined under a fluores-cent microscope. Cultivated MSCs that incubated withoutprimary antibody were considered as negative control.

2.10 In vitro migration assayThe migration assay was performed using Chemicon QCM96-well cell migration assay (8 µm pore size). About 4 � 104

cells in 100 µL were seeded in the upper chamber of theprovided 96-well plates. Different concentrations of recombi-nant rat SDF-1 and MCP-1 were provided and added to thelower chamber as attractants. The plates were incubated 6 h

at 37�C in a CO2 incubator to allow for MSCs migrationthrough the pores. The cells attached to the outside of thechamber were detached using the detachment buffer and col-lected according to manufacturer’s instructions. The cellswere lysed and stained with CyQuant GR dye. Total cellmigration was determined by measuring the optical densityof migrated cells using 480/520 nm filter set.

2.11 Animal model of diabetesDiabetes was induced in rats by a single intraperitoneal injec-tion of 65 mg/kg STZ in sodium citrate buffer. The controlgroup received saline. After 3 and 7 days, blood samples wereobtained via tail vein and glucose levels were assessed using aglucometer. Rats with a blood glucose level of > 200 mg/dLwere considered diabetic [28,29].

2.12 In vivo migrationThe ability of MSCs to migrate toward pancreas was assessedin vivo by injecting 1 � 106 MSCs labeled with CM-Dil intothe tail vein of 6- to 8-week-old rat. Diabetic animals weredivided into two groups (n = 3): group I received DFO-treated MSCs and group II received untreated MSCs.A total of 24 h after cell transplantation, all animals wereintracardially perfused with cold saline and 8% paraformalde-hyde. The pancreas was then dissected out and placed into 8%paraformaldehyde solution for at least 24 h before being proc-essed for paraffin embedding. Then tissue was embedded inparaffin. The 5 µm sections of the sample were obtained. Sec-tions were counterstained with Hoechst. Finally the numberof CM-Dil-labeledMSCs in the pancreas was assessed on serialsections under a fluorescence microscope. Data was quantifiedusing Image J software (NIH, Bethesda, MD, USA).

2.13 Statistical analysisData are presented as mean ± standard error of the mean(SEM). Unpaired Student’s t-test was performed for compar-isons between two groups and a one-way ANOVA was per-formed for multiple comparisons. A p value of < 0.05 wasconsidered to be statistically significant.

3. Results

3.1 Cell surface characterization of MSCsThe cell surface markers of MSCs were analyzed using flowcytometric assay. Results have shown that MSCs expressedCD44, CD73, CD105, CD106 and CD90, while it was neg-ative for CD11b, CD31, CD34 and CD45 (Figure 1).

3.2 Cell viabilityThe effects of increasing concentrations of DFO on MSCsviability were examined using MTT assays. As shownin Figure 2, DFO at a concentration of 150 µM for 24 h, sig-nificantly increased MSCs viability and hence this was appliedin all other experiments throughout the study.



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3.3 HIF-1a expressionTo determine the effect of DFO on HIF-1a, the proteinexpression levels of HIF-1a in MSCs were assayed by westernblotting. In a time course study, DFO treatment induced anincrease in HIF-1a protein expression in a time-dependentmanner reaching maximum at 24 h (Figure 3A). However,the elevation of HIF-1a protein by DFO was reversed by pre-treating cells with 2-ME (5 µmol). Densitometry analysisshowed that DFO significantly increased HIF-1a proteinexpression compared to untreated control group (Figure 3B).Immunocytochemical studies also confirmed significantlyhigher expression of HIF-1a (Figure 3C).

3.4 CXCR4 and CCR2 genes and protein expressionThe effects of DFO on the expression of CXCR4 andCCR2 mRNA in MSCs were examined using semiquantita-tive RT-PCR assays. Densitometric analysis showed that theCXCR4 and CCR2 expression was significantly increased inthe DFO-treated MSCs time-dependently up to 24 h com-pared to control group (Figure 4A). The overexpression ofCXCR4 and CCR2 by DFO was reversed by 2-ME2. More-over, to confirm whether the increase of mRNA for CXCR4and CCR2 were translated into the protein, CXCR4 and

CCR2 protein expression levels were measured by westernblot assay. Similar to the RT-PCR data, DFO-treated MSCsexpressed a higher (two- to threefold) level of CXCR4 andCCR2 protein compared to untreated controls (Figure 4B).

3.5 MMP-2 and MMP-9 activityThe hydrolytic activity of MMP-2 and MMP-9 were analyzedusing gelatin zymography. Noticeable increase in zymo-graphic activity was observed in treated MSCs (Figure 5A).Densitometric analysis of the lytic regions from the zymo-grams showed that DFO caused higher activity of MMP-2and MMP-9 up to 40 -- 45% compared to untreated controls(Figure 5B). The effects of DFO were inhibited by 2-ME.

3.6 In vitro migration assayThe migration ability of cells were quantified using QCMchemotaxis 96-well cell fluorometric migration assay. DFO-treated MSCs were migrated toward different concentrationsof SDF-1 and MCP-1 in a dose-dependent manner (Figure 6).Compared to untreated cells, DFO-treated MSCs for 24 hshowed a significantly higher migration capacity in responseto SDF-1 and MCP-1.

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Figure 1. Characterization of rat BM-MSC surface markers using flow cytometry. Flow cytometric analyses showed that

cultured cells were positive for CD44, CD73, CD90, CD105 and CD106. These cells were negative for CD11b, CD31, CD34 and

CD45. The respective isotype control is shown as a black line. The data are representative of three independent experiments.



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3.7 In vivo migration and homing of MSCs to

diabetic pancreasTo assess the migration of MSCs to the pancreas, CM-Dil-labeled MSCs were infused through the tail vein intoSTZ-induced diabetic rats. After 24 h, the animals were sacri-ficed and CM-Dil+ MSCs were examined by fluorescencemicroscopy in the pancreas. The results indicated that signif-icantly a higher number of grafted MSCs (~ 60%) wereobserved in rats receiving treated MSCs compared to ratsreceiving non-treated MSCs, confirming that DFO precondi-tioning could increase the homing levels of MSCs (Figure 7)(p < 0.05).

4. Discussion

Recently pharmacological preconditioning has been proposedhypothetically and experimentally as an efficient approach formodification and improvement in function of various organ,tissue and cells and in particular stem cells [19,24,25,30,31].In the present study, we investigated the effect of DFO pre-

conditioning on molecular mechanisms involved in homingof MSCs particularly with the goal of its application in celltherapy of type 1 diabetes. For this purpose, the MSCs weretreated with DFO and the expression levels of HIF-1a,CXCR4, CCR2, as well as the activity of MMP-2 andMMP-9 were evaluated. Finally, we studied the migration ofMSCs toward the damaged tissue both in vitro and in vivo.To our knowledge, these results for the first time demon-

strated that DFO improved MSCs homing through enhancingCXCR4/CCR2 expressions and MMP-2/MMP-9 activity,

which were due to accumulation and stabilization of HIF-1ain these cells.

Iron chelators such as DFO, besides its normal use in ironoverdose and poisoning, has been shown to have many othereffects both in the clinical and experimental setting. Theyhave immunomodulatory effects and antioxidant propertiesby inhibiting the formation of hydroxyl radical through theFenton or Haber--Weiss reaction. In addition, iron chelatorsare used as hypoxia-mimetic agent having the ability to inducea number of hypoxia response genes including those responsi-ble for angiogenesis, tissue regeneration and cell survival andfate [32-34]. Other evidence show that DFO treatment increaseswound healing by enhancing VEGF expression and prolifera-tion of vasculature in diabetes [35].

In addition, DFO treatment in STZ-diabetic rat has alsobeen shown to elevate antioxidative enzyme activity, loweroxidative stress markers and normalize diabetic vascularhyperresponsiveness to drugs [36].

In a transplantation study, it is indicated that pretreatmentof rat pancreatic islets with DFO stimulates VEGF expressionand enhances islet vascularization and subsequently improvestheir viability when transplanted [37]. Furthermore, precondi-tioning of encapsulated human islets with DFO stabilizesHIF-1a, increases VEGF expression and improves islets abil-ity to function better during transplantation as well as reducesgraft rejection [38]. Moreover, in cold ischemia injury, themain cause of graft dysfunction and loss, studies have shownthat DFO is able to reduce ischemia-associated renal injuryand increase renal efficiency and its function after kidneytransplantation [39]. DFO has also been shown to exert

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Figure 2. Measurement of MSCs viability by MTT assay. The MSCs were treated with different concentrations of DFO for 24,

48 and 72 h. The viability of treated cells were significantly increased compared to the control group. Culture medium

without FBS (0%) served as a negative control and medium with 15% FBS served as a positive control.Results are reported as the mean ± SEM (n = 8) *p < 0.05 versus control.



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Figure 3. Measurement of HIF-1a by western blotting and immunocytochemistry. The untreated and treated MSCs with DFO

at the concentration of 150 µM for different times (0, 8, 16 and 24 h). (A) MSCs proteins were extracted and analyzed for

HIF-1a expression by western blotting. (B) Western blotting densitometry revealed a higher expression of HIF-1a protein up to

12 and 24 h as compared with the untreated cells. These effects can be blocked by pretreatment of cells with 2-ME. Data are

represented as mean ± SEM. (n = 3), Statistically significant differences from the control group: *p < 0.05, zp < 0.001.

(C) Detection of the HIF-1a in MSCs by immunocytochemistry. MSCs were incubated in the HIF-1a antibody followed by

incubation with FITC-conjugated secondary antibody (green). Nuclei were counterstained with DAPI (blue). Immunocyto-

chemistry analysis of HIF-1a expressions showed DFO-induced nuclear expression of HIF-1a in MSCs and fluorescence signal

were intensified in DFO-treated MSCs compared to untreated controls.



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neuroprotective effects in cerebral ischemia and decreasesbrain damage by induction of HIF-1a and erythropoietinexpression [33,40].One of the most important proposed mechanisms for the

therapeutic use of DFO is the stabilization of HIF-1a [32].PHDs are recognized as the main regulators for HIF-1a activ-ity that act via regulation of ubiquitination and subsequentdegradation of the HIF-1a subunit in proteasome and DFOcan block HIF-1a degradation by chelating iron an essentialcofactor for PHDs activity [41,42]. It is important to realizethat HIF-1 dysfunction plays an important role in diabetesbut involved complications.

The current study showed that treatment of the MSCs withDFO stabilizes HIF-1a protein. These results are consistentwith previous reports that several non-hypoxic agents suchas DFO can stabilize HIF-1a in neural progenitor (NPG)cells [43]. In this study it has also been observed thatCXCR4 and CCR2 are constitutively expressed by MSCsand treatment of the cells with DFO significantly increasedtheir mRNA as well as protein expressions. In order to inves-tigate HIF-1a mediated effect of DFO on CXCR4 andCCR2 expression, the expression levels of these cytokinereceptors were measured in presence of HIF-1 blocker,2-ME. It has been reported that 2-ME could block HIF-1a

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Figure 4. mRNA and protein expressions measurement of chemokine receptors, CXCR4 and CCR2. In MSCs treated with

150 µM DFO for 0, 8, 16 and 24 h, the expression of CXCR4 and CCR2 were significantly increased compared to untreated

controls. (A) Total RNA was analyzed by RT-PCR for CXCR4 and CCR2 mRNA expressions. DFO-treated MSCs expressed higher

levels of CXCR4 and CCR2 mRNA in time-dependent manner compared to untreated group, *p < 0.05, zp < 0.01,§p < 0.001 versus non-treated cells. CXCR4 and CCR2 mRNA induction expression were inhibited by 2-ME2. b-Actin was used as

loading control. (B) CXCR4 and CCR2 protein levels were analyzed by western blot. Data showed that DFO, markedly elevated

the CXCR4 and CCR2 protein expression, *p < 0.001 versus untreated cells (n = 3).



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protein synthesis as well as inhibite its transcriptional activ-ity [44]. 2-ME treatment reversed the DFO-mediated increasesof these cytokine receptors expression. These findings indicatethat HIF-1a plays an essential role in induction of CXCR4and CCR2 expression. These results are similar to previousstudies indicating that HIF-1a can induce CXCR4 upregula-tion in various cell types, such as ECs, macrophages, monocytesand cancer cells [45,46].

Further, our study indicated that preconditioning of MSCswith DFO increases the activity of MMP-2 and MMP-9which was reversed by HIF-1a inhibitor. Elevation of proteo-lytic enzymes such as MMP-2 and MMP-9 are essential forcell migration and homing by degradation of ECM enablingcells to pass through barrier and reach target tissue [47]. Thisfinding implies that the increase of DFO in these proteasesactivity is also regulated by HIF-1a. This finding is in agree-ment with results reported by Lolmede in which treatment ofthe cells by hypoxia mimetic agents increased MMP-2 and

MMP-9 activity in the adipocyte [48]. In contrast, these resultswere in opposite to the study published by Jin et al. [49].reporting that treatment of the hepatic satellite cells withDFO caused a decreased mRNA expression of MMP-2 andMMP-9. A possible explanation for this might be due to var-iation in the sources of the cells. It has also been reported that,MT1-MMP, the MMP-2 physiological activator, has twoputative HIF-binding sites which are activated under hypoxicconditions causing subsequent activation of MMP-2 [50]. Thismay, therefore, be similarly activated by DFO-inducedHIF-1a stabilization in the current study.

To confirm this in vitro improvement of homing mecha-nisms, we utilized the in vitro migration assay, followedby in vivo study in diabetic rats. We analyzed the in vitromigration capacity of the cultured MSCs toward two chemo-attractive chemokines, SDF-1 and MCP-1, using transwellmigration assay. MSCs treatment with DFO resulted inhigher migration of these cells compared to untreated

– – + DFO2-ME

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Figure 5. Representative gelatin zymograms of MMP-2 and MMP-9 activity in MSCs. (A) MMP-2 and MMP-9 activity is

enhanced by DFO. (B) Quantitative analysis was performed with densitometry. These effects can be blocked by preincubating

cells with 2-ME.*p < 0.05, zp < 0.01 versus untreated cells (n = 3).



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MSCs. This result may be explained by the fact that DFO ele-vate the expression of CXCR4 and CCR2, the receptors forSDF-1 and MCP-1 chemokines which were thought to beupregulated in damaged tissue, and hence facilitated themigration of MSCs to the site of injury.The effects of MCP-1 are shown to be exerted by binding

to CCR2, a G-protein coupled receptor, that is activated bychemokine ligands (CCL), including MCP-1/CCL2, CCL7,CCL8, CCL12 and CCL13. Activation of intracellular effec-tors of CCR2 such as adenylate cyclases, PLCbs, PI3Ks,MAPKs and Rho GEFs occurs mostly through heterotrimericG-proteins of the GI/O and the Gq family [51]. It has beenshown that on MCP-1 stimulation, CCR2 binds to the adap-tor molecule FROUNT and induces polarization of stem cellsthrough the PI3K-Rac pathway, resulting in progression ofstem cell migration [15]. The current results are similar toEPCs expressing CCR2 receptor on their surface mediatingchemotaxis to the area of endothelial denudation whichsecrete MCP-1/CCL2 and SDF-1 causing angiogenesis [52].Similarly, CCR2 is also shown to be expressed on the surfaceof NPGs, and it is shown that in response to neuroinflamma-tion stimuli, increased MCP-1 expression causes the migrationof NPGs [15,53]. Interestingly, this pattern shows that inmalignant gliomas, which express chemokines such as MCP-1and SDF-1, mediating MSCs migrate toward tumor [54]. It isexpected, therefore, that increased CCR2 expression in DFO-treated MSCs exerts an enhancing role for migration and hom-ing of MSCs toward the MCP-1 secreted by diabetic injuredpancreas in vivo and MCP-1 used in in vitro chamber. It mayalso be speculated that in clinical setting, since the expressionof MCP-1 from pancreatic islets, which plays an importantrole in the induction of autoimmune response that eventually

leads to type 1 diabetes in human, could attract more DFO-preconditioned MSCs overexpressing CCR2.

Finally, we investigated the role of DFO on migration andhoming of Dil-labeled MSCs toward the pancreas in diabeticrat in vivo. About 24 h after injection of MSCs, fluorescencestaining showed increased numbers of Dil-labeled cells withinthe pancreas in DFO-treated cells compared to untreated cells.

To explain this in vivo effect of DFO, it is believed that indamaged and inflamed tissue concentration of inflammatorycytokine increases causing migration and homing of MSCsto injured site in vivo in a chemotactic manner [47]. It is alsoknown that MSCs home in response to SDF-1 which isreleased by injured tissue [55-57]. In our study, therefore, itcould be explained that CXCR4-overexpressed MSCs treatedwith DFO would be chemoattracted toward its ligandSDF-1 released by STZ-induced injured pancreas of diabeticrats. This is possibly in a similar pattern with results wehave shown in in vitro migration assay, using SDF-1. This isconsistent with what has been shown in the damaged myocar-dium in which the concentration of plasma SDF-1 increaseslead to enhanced recruitment of stem cell into the injuredmyocardium [58].

To investigate the mechanism of SDF-1, some studies haveshown that the effect of SDF-1 on progenitor cell migration ismediated in part via PI3K/Akt/endothelial nitric oxide syn-thase (eNOS) pathway [59]. It has also been shown thateNOS and SDF-1 genes are regulated by HIF-1a [18]. In theischemic myocardium, eNOS increases SDF-1 expression viacGMP and in turn interaction of SDF-1a and CXCR4, lead-ing to MSCs homing toward myocardium [60]. Observationsshow that the effect of SDF-1 on migration of progenitorcell is blocked by the Akt and NOS inhibitors [61].

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100 ng/mlMCP-1

∗

∗

∗

∗

Figure 6. Measurement of the effect of DFO on in vitro migration of MSCs. MSCs were treated with or without DFO for 24 h

and then their ability for migration toward various concentrations of SDF-1 and MCP-1 were evaluated. DFO significantly

increased MSCs migration toward SDF-1 and MCP-1. The maximum effect was observed at concentrations of 500 ng/mL of

SDF-1 and MCP-1. Control wells contain only cells without a chemoattractant. Samples without cells, but containing cell

detachment buffer, lysis buffer and CyQuant Dye were typically used as ‘blanks’ for interpretation of data. Each condition was

performed in triplicate.*p < 0.05 versus untreated cells (n = 3).



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Taken together, the current data indicated that DFO byaffecting factors involved in migration and homing of MSCssuch as chemokine receptors and proteolytic enzyme couldenhance the number of MSCs reaching to target tissue.

However, to complete this study, further studies are stillrequired to understand the precise and detailed mechanismunderlying the effect of DFO on the homing signaling ofMSCs. Also, despite fact that this study was focused on

MSC homing and the mechanisms involved in this process,it would have been more complete if glucose and insulin levelswere measured after DFO-treated MSCs transplantation intodiabetic rat and investigating the ability of these cell trans-plantation to restore normal conditions.

In conclusion, these results performed in vitro and in vivo,for the first time indicating that the survival, proliferation,migration and homing potential of MSCs which are delivered

100µm 100µm 100µm

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Figure 7. Measurement of MSCs migration to pancreas using in vivo migration assay. DFO increasedMSCs migration in vivo.

(A) Representative images of MSCs implantation in the pancreas (red). Counterstaining was performed with HOECHST (blue). (B)

Representation of average number of migrated DFO-treated and untreated MSCs. Comparing the two, it can be seen that in

diabetic ratswhich receivedDFO-treatedMSCs, thegreaterpercentageofcellsweremigrated to thepancreas compared todiabetic

rats receiving non-treated MSCs. Data shown are representative of three individual experiments for each group.*p < 0.05.



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for therapeutic purpose, were significantly affected by DFO.These results may also have clinical implications in which pre-treatment and preconditioning of MSCs with DFO improvesthe tissue regenerative and therapeutic potential in order toenhance efficacy of cell therapy in diabetes.

Acknowledgments

The authors especially thank M Najafi, Department ofBiochemistry, Tehran University of Medical sciences and

MA Edalatmanesh, Department of Physiology, Azad Univer-sity of Fars for valuable assistance.

Declaration of interest

The authors declare that there is no duality of interest associ-ated with this manuscript. Both authors contributed to studythe design, analysis and interpretation of data, drafting thearticle and approved the final version for publication. Thisstudy was supported by a grant from the Tehran Universityof Medical Sciences.

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AffiliationR Najafi1,2,3 & Ali M Sharifi†1,2,3,4,5

†Author for correspondence1Tehran University of Medical Sciences,

School of Medicine,

Razi Drug Research Center,

Department of Pharmacology,

Tehran, Iran2Tehran University of Medical Sciences,

School of Advanced Technologies in Medicine,

Department of Molecular Medicine,


Cellular and Molecular Research Center,


School of Advanced Sciences in Medicine,

Department of Tissue Engineering

and Cell Therapy, Tehran, Iran5Tehran University of Medical sciences,

Shariati Hospital,

Endocrine and Metabolism Research Institute,

Tehran, Iran

E-mail: [email protected]



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Deferoxamine preconditioning potentiates mesenchymal stem cell homing in vitro ...

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