Mesenchymal stem cells effectively reduce surgically induced stenosis in rat carotids

ORIGINAL ARTICLE 789J o u r n a l o fJ o u r n a l o f

CellularPhysiologyCellularPhysiology
Mesenchymal Stem Cells
Effectively Reduce SurgicallyInduced Stenosis in Rat Carotids
AMALIA FORTE,1 MAURO FINICELLI,1 MONICA MATTIA,1 LIBERATO BERRINO,1
FRANCESCO ROSSI,1 MARISA DE FEO,2 MAURIZIO COTRUFO,2 MARILENA CIPOLLARO,1

ANTONINO CASCINO,1 AND UMBERTO GALDERISI1*1Excellence Research Center for Cardiovascular Diseases, Department of Experimental Medicine,

Second University of Naples, Naples, Italy2Excellence Research Center for Cardiovascular Diseases, Department of Cardiothoracic and Respiratory Sciences,

Second University of Naples, Naples, Italy

Restenosis following vascular injury remains a pressing clinical problem. Mesenchymal stem cells (MSCs) promise as a main actor ofcell-based therapeutic strategies. The possible therapeutic role of MSCs in vascular stenosis in vivo has been poorly investigated so far. Wetested the effectiveness of allogenic bone marrow-derived MSCs in reduction of stenosis in a model of rat carotid arteriotomy. MSCswere expanded in vitro retaining their proliferative and differentiation potentiality. MSCs were able to differentiate into adipocyte andosteocyte mesenchymal lineage cells, retained specific antigens CD73, CD90, and CD105, expressed smooth muscle alpha-actin,were mainly in proliferative phase of cell cycle and showed limited senescence. WKY rats were submitted to carotid arteriotomy and tovenous administration with 5� 106 MSCs. MSCs in vivo homed in injured carotids since 3 days after arteriotomy but not in contralateraluninjured carotids. Lumen area in MSC-treated carotids was 36% greater than in control arteries (P¼ 0.016) and inward remodeling waslimited in MSC-treated carotids (P¼ 0.030) 30 days after arteriotomy. MSC treatment affected the expression level ofinflammation-related genes, inducing a decrease of IL-1b and Mcp-1 and an increase of TGF-b in injured carotids at 3 and 7 days afterarteriotomy (P< 0.05). Taken together, these results indicate that allogenic MSC administration limits stenosis in injured rat carotids andplays a local immunomodulatory action.

J. Cell. Physiol. 217: 789–799, 2008. � 2008 Wiley-Liss, Inc.

To the memory of Prof. Antonino Cascino, deceased 6 April 2008.

Contract grant sponsor: SHRO 2007–2008 ‘‘Role of cell cycle-related genes in the biology of stem cells’’.Contract grant sponsor: Legge 5 Regione Campania 2003 ‘‘Modellosperimentale di iperplasia fibrointimale post-chirurgica in modelli diratto affetti da patologie dell’apparato cardiovascolare’’.Contract grant sponsor: Progetto Finalizzato Sanita 2003‘‘Patologie infettive e insulto chirurgico: studi di genomica eproteomica nel remodeling vascolare’’.

*Correspondence to: Umberto Galderisi, Department ofExperimental Medicine, Section of Biotechnology and MolecularBiology, Second University of Naples, Via L. De Crecchio,7—80138 Naples, Italy. E-mail: [email protected]

Received 21 April 2008; Accepted 11 July 2008

Published online in Wiley InterScience(www.interscience.wiley.com.), 8 August 2008.DOI: 10.1002/jcp.21559

Restenosis is a pathophysiological phenomenon that can occurin patients submitted to revascularization procedures (bypass,endartherectomy, angioplasty), possibly resulting in newnarrowing of injured vessels. Vascular restenosis remains apressing clinical problem, despite the therapeutic strategies anddevices developed so far (Lal, 2007).

The disruption of endothelial cells (ECs) in the intima byvascular injury is of particular importance in stenosisprogression, since it is the cause of a concomitant reduction ofvasculoprotective mediators (e.g., nitric oxide andprostacyclin). EC disruption also triggers a number of signalingcascades that lead to inflammation, platelet adhesion and cellproliferation. Also the intraoperative damage to adventitia andto vasa vasorum and nervous fibers it contains can havedeleterious effects on restenosis (Khurana et al., 2004).

Stem cells hold a great potential for the regeneration ofdamaged tissues in cardiovascular diseases. Recent studiesclearly indicated that different bone marrow-derived stem cellpopulations, characterized by different markers and withdistinct behaviors, contribute to vascular remodeling afterinjury. Nevertheless, the exact role of vascular cell precursorsin restenosis pathophysiology is not yet well defined, asheterogeneous and contrasting data are currently available(Tanaka et al., 2003; Forte et al., 2007a), probably in relation tothe different kind of injury, to the animal models and to theheterogeneity of stem cell populations.

Mesenchymal stem cells (MSCs) are non-hematopoieticmulti-potent stem-like cells able of differentiating into bothmesenchymal and non-mesenchymal lineages. MSCs offer aseries of advantages: (a) they can be isolated from a smallaspirate of bone marrow; (b) extensively proliferate in vitrowhile preserving a normal karyotype and telomerase activity onseveral passages (Pittenger et al., 1999); (c) express lowimmunogenicity (Beggs et al., 2006), and hence their use in

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animal models should not require a pharmacologicalimmunosuppression.

MSCs have an intrinsic ability to differentiate into functionalcell types able to repair the diseased or injured tissue in whichthey are localized. This trend to adopt the local cellular identitymay be correlated to tissue cytokines and matrix factors, as wellas to adequate contact with host cells. For this reason, MSCsare currently under scrutiny for treatment of differentcardiovascular diseases. In the concern of heart valve surgery,for example, tissue-engineered porcine heart valves createdfrom MSCs in a lamb model, injected in a decellularizedxenograft scaffold, exhibited satisfactory hemodynamic andhistological aspects (Vincentelli et al., 2007).

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It has not yet been clearly determined whether MSCs cansubstantially contribute to a positive resolution of restenosis aftervascular injury. As a matter of fact, limited and contrasting resultsare currently available in experimental models. In more detail,Chen et al. (2007) revealed in rats submitted to aortic angioplastythat the local infusion of MSCs induced higher restenosis thancontrols. Another study also linked the homing of MSCs to anexcessive repair process of the vessel in a model of mice femoralartery wire injury (Wang et al., 2008). Conversely, an ex vivomodel of endothelium repair developed in rabbit revealed thepositive role played by MSCs in prevention of smooth muscle cell(SMC) proliferation and in differentiation to ECs (Wu et al., 2005).Moreover, MSCs have been successfully applied in engineeredvascular grafts, showing excellent long-term patency (Hashi et al.,2007). Finally, a recent study demonstrated that MSCs seeded onpolyurethane patch restore endothelium in rat aorta and inducecomplete SMC differentiation, restoring a media-like thick wall(Mirza et al., 2008).

MSCs are able to suppress both B and T cell function(Rasmusson et al., 2003; Corcione et al., 2006). Allogenic MSCssuppress T cells activation by increasing the CD8(þ)subpopulation and decreasing the CD25(þ) T lymphocytesubset. One of the major mediators of T-cell suppression byMSCs is their production of nitric oxide (NO) by inducible NOsynthase, that is not induced in T-cells (Sato et al., 2007). Thisobservation can have potential positive repercussions onapplication of MSCs for restenosis reduction, since it has beendemonstrated that inflammation plays a major role inrestenosis, as lymphocytes recruited at the injury site secretegrowth factors that activate vascular cell proliferation.Moreover, NO production by MSCs can have positive effectson vascular remodeling, since it is a mediator of vasorelaxationand has effects on the modulation of the SMC phenotype,maintaining them in a contractile status. MSCs are known toinhibit the response of allogeneic T lymphocytes in vitrothrough different pathways. Two different mechanisms, eithercell contact-dependent or independent, have been proposed toaccount for this immunosuppression (Chen et al., 2007), basedon the expression of soluble factors, including IL-10 and TGF-b,after contact with T lymphocytes (Nasef et al., 2007). Theseobservations imply that allogenic MSC transplantation may beaccomplished without the need for host immunosuppression.

In this study we aimed to verify the role, positive or negative,played by MSCs in stenosis progression induced by a previouslycharacterized model of rat vascular injury based on carotidarteriotomy (Forte et al., 2001). We demonstrated that asystemic administration of allogenic bone marrow-derivedMSCs injected via tail vein are able to home at the injury site andto limit lumen stenosis. Our data also reveal that MSCs are ableto affect the local expression of inflammation-related genes.These findings add new information about the possibletherapeutic role in restenosis for this peculiar population ofstem cells.

MethodsAnimals

Studies were carried out on 12-week-old male Wistar Kyoto(WKY) rats (230–250 g) (Charles Rivers, France). All animals werehandled in compliance with the ‘‘Ethical principles and guidelinesfor scientific experiments on animals’’ of the Swiss Academy ofMedical Sciences. All protocols were approved by the Animal Careand Use Committee of the Second University of Naples. Rats wereacclimatized and quarantined for at least one week beforeundergoing surgery.

MSC cultures

MSCs have been harvested from the bone marrow of the femursand tibias of adult WKY rats. WKY rats have been anaesthetized

JOURNAL OF CELLULAR PHYSIOLOGY

with an intraperitoneal injection of ketamine hydrochloride andMSCs have been harvested from the bone marrow by inserting a21-gauge needle into the shaft of the bone and flushing it withcomplete a-modified Eagle’s medium (aMEM) containing 20% fetalbovine serum (FBS), 2 mM L-glutamine, 100 U/ml penicillin, 100mg/ml streptomycin. Cells from one rat have been plated into two100 mm dishes. After 24 h, non-adherent cells have been discarded,and adherent cells have been washed twice with phosphatebuffered saline (PBS). The cells have been then incubated for5–7 days to reach confluence. Finally, MSCs have been extensivelypropagated for further experiments. All cell culture reagents havebeen obtained from Invitrogen (Milan, Italy).

MSC differentiation

Differentiation of isolated MSCs into the mesenchymalosteogenetic and adipogenetic lineages was induced using differentprotocols.

Adipocyte differentiation: cells were grown for 14 days inaMEMcontaining 10% FBS, 100 mM indomethacin, 1 mM hydrocortisone,1 mg/ml insulin, and 10% horse serum. Adipocytes were detectedby standard Oil Red-O staining.

Osteocyte differentiation: cells were grown for 14 days inaMEM containing 10% FBS, 100 mM dexamethasone, 0.2 mMascorbic acid, and 10 mM b-glycerophosphate. Osteocytes weredetected with Alizarin Red staining.

Senescence-associated b-galactosidase staining

Cultured MSCs were fixed for 10 min with 2% formaldehyde/0.2%glutaraldehyde at room temperature (RT). Cells were then washedtwice with PBS and 2 ml of senescence-associated b-galactosidasestaining solution per 35 mm dish were added. Cells were incubatedat 378C until blue staining was clearly detectable under a lightmicroscope. The percentage of senescent cells was calculated bythe number of b-galactosidase positive cells out of at least 500 cellsin different microscope fields. Just before using thesenescence-associated b-galactosidase staining solution, we added20 ml of citric acid/sodium phosphate in a volume of 100 ml, pH 6.0,150 mM NaCl, 2 mM MgCl2, 5 mM potassium ferricyanide, 5 mMpotassium ferrocyanide and 1 mg/ml X-Gal.

TRAP assay

The telomere repeat amplification protocol (TRAP) assay wasperformed according to Kim and Wu (1997). Briefly, 5� 105 MSCsor control HEK cells were lysed in 10 mM Tris–HCl, pH 7.5, 2.5 mMMgCl2, 1 mM EGTA, 0.5% CHAPS, 10% glycerol, 5 mMb-mercaptoethanol, and 1 mM AEBSF for 30 min at 48C. Thelysates were then centrifuged for 10 min at 10,000g at 48C. Aftercentrifugation, protein concentration was determined by theBradford assay. The reaction mixture for the TRAP assay was doneas follows: 1� Taq DNA polymerase buffer (Promega, Madison,WI), 1.5 mM MgCl2, 50 mM dNTPs, primer M2(TS) (50-AATCCG-TCGAGCAGAGTT-30) and TRAP internal control (50AATCCG-TCGAGCAGAGTTAAAAGGCCGAGAAGCGAT-30). Thismixture was incubated for 30 min at 258C; we then added primerACX (50-GCGCGG[CTTACC]3CTAACC-30), primer NT (50-ATCGCTTCTCGGCCTTTT-30) and 2.5 U Taq DNA polymerase(Promega). The reaction was denatured for 3 min at 948C, thenamplified for 30–35 cycles (948C for 15 sec, 608C for 15 sec, 728Cfor 15 sec). Primers ACX and M2(TS) amplify the telomeraseproducts, whereas primers M2(TS) and NT amplify the TRAPinternal control. PCR products were resolved on 20%polyacrylamide gels stained with 1� GelStar nucleic acid stain(Lonza, Basel, Switzerland).

Cell cycle analysis

For each assay, 3� 105 MSCs were collected and resuspended in500 ml of a hypotonic buffer (0.1% Triton X-100, 0.1% sodiumcitrate and 50 mg/ml propidium iodide/RNAse A). Cells were

M S C s R E D U C E V A S C U L A R S T E N O S I S 791

incubated in the dark for 30 min and then analyzed. Samples wereacquired on a FACSCalibur flow cytometer using the Cell Questsoftware (Becton Dickinson, Fanklin Lakes, NJ) and analyzed usingthe Cell Quest software and the ModFitLT software version 3(Becton Dickinson).

Fluorescence immunocytochemistry

MSCs (1.5� 104) were used for immunofluorescence analysis ofthe expression of surface antigens CD73, CD90, and CD105, andof smooth muscle (SM) alpha-actin. Cultured MSCs grown in slidechambers (BD Falcon) were fixed in 4% buffered paraformaldehydefor 10 min at RT, incubated with blocking solution (1% BSA) for 1 hand then with the primary antibody (mouse anti-rat CD73, BDBiosciences, San Jose, CA, 1:100; mouse anti-rat CD90, BDBiosciences 1:250; goat anti-rat CD105, Santa Cruz, Santa Cruz,CA, 1:100; mouse anti-rat SM-alpha actin, Sigma-Aldrich, Milan,Italy, 1:250) in blocking solution overnight at 48C. Negativecontrols were incubated with blocking solution only. Subsequently,the samples were incubated with monoclonal anti-mouse oranti-goat FITC- or TRITC-conjugated secondary antibodies(dil 1:100, Jackson ImmunoResearch, Suffolk, UK) for 1 h at RT. Thenuclei were stained with Hoechst 33258 (Sigma-Aldrich) for 5 minand the samples examined under a fluorescence microscope (Leica,Wetzlar, Germany).

RNA extraction and RT-PCR analysis

Total RNA was extracted from cultured MSCs, from injured ratcarotids at 3 and 7 days after arteriotomy (n¼ 4 DiI-MSC-treatedrats and n¼ 4 DiI-DMEM-treated rats, for each time point) andfrom uninjured rat carotids (n¼ 3) using the RNAeasy minikit(Qiagen, Hilden, Germany) according to manufacturer’sinstructions. RNA was treated with DNase (Qiagen) to removeDNA contamination. RNA concentration was measured using aNanoDrop ND-1000 spectrophotometer (NanoDropTechnologies, Wilmington, DE). RNA integrity was verified byelectrophoresis on denaturing 1% agarose gel. Absence of residualDNA was verified by PCR on total RNA without reversetranscription.

cDNA was generated from 200 ng of each RNA sample. Reversetranscription was done at 428C for 1 h in presence of randomexamers and Moloney-Murine Leukemia Virus (M-MULV) reversetranscriptase (Finnzymes, Espoo, Finland). GeneBank sequencesfor rat mRNAs and the Primer Express software (AppliedBiosystem) were used to design primer pairs for the genes PPAR-g,osteopontin, IL-6, IL-1a, IL-1b, TGF-b, TNF-a, IFN-g, Mcp-1,SM22, vWF and the house keeping gene GAPDH. Primersequences are available upon request.

Primer pairs were chosen to yield 100–150 bp PCR productsand were validated running the PCR products on agarose gel toconfirm a single band. Melting curves (65–948C) were alsogenerated to determine whether there were any spuriousamplification products. Each RT-PCR reaction was repeated atleast three times. A semi-quantitative analysis of mRNA levels wasperformed by the GEL DOC UV system (Bio-Rad, Hercules, CA)on agarose gels. When minimal differences in gene expression weredetected, experiments were repeated using the real-time PCRassays, run on an Opticon 4 machine (Bio-Rad). Reactions wereperformed according to the manufacturer’s instructions usingthe SYBR Green PCR master mix (Stratagene, La Jolla, CA).Relative quantitative RT-PCR was used to determine the folddifference for genes. The real time PCR efficiency was calculatedfor each primer pair using a dilution series and the MJ Opticon IIanalysis software.

Vascular injury and MSC treatment

Arteriotomy of rat common carotid artery was performed asalready published (Forte et al., 2001). Briefly, a plastic Scanlomclamp for coronary artery grafting was placed for 10 sec on the


carotid causing a crushing lesion to the vessel. At the same pointwhere the clamp was applied, a 0.5 mm longitudinal incision wasdone on the full thickness of the carotid. The incision did not crossto the other side of the vessel. Haemostasis was obtained with asingle adventitial 8.0-gauge polypropylene stitch. Once bleedingstopped, the carotid artery was carefully examined and bloodpulsation was checked distally to the incision.

For morphometric analysis, WKY rats submitted toarteriotomy were administered with 5� 106 MSCs resuspended in200ml DMEM via tail vein injection (n¼ 8), while control rats wereadministered with 200 ml DMEM (n¼ 8). For homing analysis,5� 106 MSCs were labeled with fluorescent Vybrant DiI(Molecular Probes-Invitrogen), according to manufacturer’sinstructions, and were administered to rats with 200 ml DMEM(n¼ 3 for each time point) while control rats were administeredwith 200 ml DiI-DMEM (n¼ 3 for each time point).

Histological analysis

Carotid arteries were harvested 30 days after arteriotomy andMSC administration for morphological and morphometric analysis.Alternatively, carotid arteries were harvested at 3 and 7 days afterarteriotomy for analysis of MSC homing at the injury site.Harvested vessels were fixed in 4% buffered formaldehyde,dehydrated and embedded in paraffin. Five micrometerscross-sections were stained with hematoxylin-orcein for nucleusand elastic fiber staining, respectively. Image screening andphotography of serial cross-sections were performed using a LeicaIM1000 System. Arterial remodeling, lumen cross-sectional area(CSA) and the CSA of the tunica mediaþ the intima (Mþ I) weremeasured using the Leica IM1000 software. Arterial remodeling isdefined as a change in arterial size compared with a control artery.In agreement with other studies (Hollestelle et al., 2004), we usedthe external elastic lamina (EEL) area calculated from the EEL lengthas a measure of arterial size. Total Mþ I CSA was measuredbetween the EEL and the lumen. The lumen and Mþ I CSA of eachinjured carotid were compared both to the ipsilateral distal regionand to contralateral uninjured carotid. Measurements wereperformed by two independent observers.

For analysis of DiI-labeled MSC homing at the injury site, carotidcross-sections were stained with fluorescent dye Hoechst 33258(Sigma-Aldrich) for nuclear identification. Image screening andphotography of serial cross-sections were then performed usingthe Leica 4000F software.

Statistical analysis

All statistical analysis was performed using GraphPad software(Prism 4.0). Data are presented as the mean� SEM. Statisticalsignificance was determined using two-way analysis of variancefollowed by Bonferroni’s multiple comparison test. Values ofP< 0.05 were considered significant.

ResultsCultured bone-marrow-derived rat MSCs are able todifferentiate to adipocyte and osteocyte phenotypes

One of the minimal criteria to identify multipotent MSCs is theircapacity for mesenchymal lineage differentiation (Dominiciet al., 2006). Before using MSCs in vivo to test their ability inlimiting arterial stenosis, we verified that the MSCs weextracted from rat bone marrow and expanded in vitro for15 days from passage zero (Fig. 1A) were able to differentiate toosteocytes and adipocytes using standard in vitroculture-differentiating conditions.

Osteogenic differentiation of MSCs was accompanied bymorphological changes in cells, by positive staining with AlizarinRed (Fig. 1B) and by the increased expression of the specificmarker osteopontin, determined by RT-PCR (Fig. 1D).

Fig. 1. Bone marrow-derived MSCs are able to differentiate to adipocyte and osteocyte phenotypes. A–C: Representative micrographs of ratbone marrow MSCs induced to differentiate into mesenchymal lineages with different media. MSCs in conventional culture (A); MSCs induced todifferentiatetoosteocytesandstainedwithAlizarinRed(B);MSCs inducedtodifferentiatetoadipocytesandstainedwithOil redO(C).D:PPAR-gandosteopontinmRNAanalysisbyRT-PCRontotalRNAextractedfromMSCsinconventionalcultureandinducedtodifferentiateintoosteocytesand adipocytes with different media.

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Adipogenic differentiation of MSCs was accompanied bymorphological changes in cells, by the formation of lipidvacuoles, visualized by staining with Oil Red O (Fig. 1C), and bythe increased expression of the specific marker PPAR-g,determined by RT-PCR (Fig. 1D).

Cultured bone marrow-derived rat MSCs are positivefor specific antigens

We verified on MSCs before their injection in vivo theexpression of markers recognized as one of the criteria toidentify MSCs (Dominici et al., 2006). In particular, wesuccessfully verified by immunocytochemistry that bonemarrow-derived MSCs expressed the surface antigens CD73(also known as ecto 50 nucleotidase), CD90 (also known asThy-1) and CD105 (also known as endoglin, a TGF-b receptorsubunit) (Fig. 2B–D).

Moreover, we successfully verified that MSCs were positivefor SM-alpha actin, another defining feature of MSCs (Fig. 2A)(Ball et al., 2007).

Cultured bone-marrow-derived rat MSCs are mainlyin the G1/S phase of cell cycle and show limitedsenescence at the time of injection in vivo

In order to obtain the sufficient amount of cells to be injected inrats at the time of arteriotomy, MSCs were cultured for 23 days,including 15 days from passage zero. Nevertheless, during thisperiod, MSCs maintained their proliferative capacity and weremainly in G1 and S phase of cell cycle (50% and 49%,respectively), as demonstrated by the FACS analysis (Fig. 3A).

Since senescence can hamper cell differentiative ability andstemness, we performed a time-dependent follow-up of cellularsenescence both through TRAP assay and in situ b-galactosidase assay.


TRAP assay provides indication about telomere shortening, amarker for cellular senescence. Telomerase activity ismeasured through a primer extension assay in whichtelomerase reverse transcriptase (TERT) synthesizes telomericrepeats onto oligonucleotide primers.

b-galactosidase assay is based on the increase in thelysosomal compartment of senescent cells of the expression ofb-galactosidase, detected as blue perinuclear stainingspecifically at acid pH.

Both these assays demonstrated that MSCs preserved theirtelomerase activity in comparison to control HEK cells (Fig. 3B)and showed a limited 23% of senescent cells at the time ofinjection in vivo (Fig. 3C).

Finally, the FACS analysis revealed suitable conditions of cellculture, as there was no appreciable peak corresponding to pre-G1 phase of cell cycle (Fig. 3A).

MSCs home at the injury site inarteriotomy-injured carotids

Carotids were harvested from WKY rats at 3 and 7 days afterarteriotomy and DiI-MSC injection to verify the homing ofMSCs at the injury site. Histological analysis conducted oncarotid cross-sections at level of injury site revealed aprogressive accumulation of DiI-MSCs in the adventitia and inperivascular tissue, more evident at 7 days after injury(Fig. 4B,C,F,G), when some DiI-MSCs were present also in thetunica media (Fig. 4D) and in the endothelium (Fig. 4H). Inparticular, DiI-MSCs were present around the polypropylenestitch applied after longitudinal incision of the vascular wall,where reparative phenomena occurred, and in the perivasculartissue. No DiI-MSCs were detectable in contralateral uninjuredcarotids, thus revealing the specificity of MSC engraftment atthe injury site (Fig. 4A,E).

Fig. 2. MSCs express lineage-specific antigens at the time of injection in vivo. Representative photomicrographs of MSCs showingimmunocytochemistry for SM-alpha actin (A), CD73 (B), CD90 (C), and CD105 (D). The cell nuclei were counterstained with Hoechst 33258 andemitted blue fluorescence. The cells emitting green fluorescence were positive for SM-alpha actin (A); the cells emitting red fluorescence werepositive for CD73 (B), CD90 (C) and CD105/Endoglin (D). Scale bar represents 50 mm.


MSCs are able to reduce arteriotomy-inducedcarotid (re)stenosis

Rat common carotids were submitted to arteriotomy andadministered with 5� 106 MSCs (n¼ 8) or with DMEM (n¼ 8)soon after carotid arteriotomy and skin suture.

Fig. 3. MSCs are in the G1/S phase of cell cycle and show limited senescenculture from passage zero (A). Polyacrylamide gel electrophoresis of TRAculture from passage zero and of control HEK cells. Each assay has been rThe lower bandsrepresent the internal control ofassay (B).Senescence-assfrom passage zero, when about 23% of senescent cells were present. Arro


The morphological and morphometric analysis of leftcommon carotid arteries was performed 30 days afterarteriotomy and treatment with MSCs or DMEM. Each carotidwas compared to its contralateral uninjured artery and to distalipsilateral uninjured region. Qualitative morphological analysisrevealed that the surgical injury induced marked neoadventitia

ce at the time of injection in vivo: FACS analysis of MSCs at 15 days ofP assay products obtained from protein extracts of MSCs at 15 days ofepeated twice. Black arrows indicate the amplification products.ociatedb-galactosidaseassayperformedonMSCs at15daysof culturew indicates a representative blue stained senescent MSC (C).

Fig. 4. MSCs home at the injury site after arteriotomy.Representative cross-sections of carotids from DiI-labeledMSC-treated rats harvested at 3 and 7 days after arteriotomy.Uninjured carotid (A,E). DiI-MSC-treated rat carotid harvested at3 days after arteriotomy (B,F). DiI-MSC-treated rat carotid harvestedat 7 days after arteriotomy (C,G). Representative DiI-MSCs in thetunica media at 7 days after arteriotomy (D). RepresentativeDiI-MSCs in the endothelium at 7 days after arteriotomy (H).Subparts (E–G) represent 40T magnification of the area enclosed inthe white perimeter in A–C (20T magnification). D–H: 40Tmagnification. Representative DiI-labeled MSCs emitting redfluorescence are indicated by small white arrows. Red arrows indicatethe point in the adventitia where the suture polypropilene stitch wasapplied after arteriotomy. The cell nuclei were counterstained withHoechst 33258 and emitted blue fluorescence. Scale bar in (D–H)represents 50 mm.

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formation along with extracellular matrix and elastic laminaaccumulation in both groups of rats, with only limited focalneointima in a few cases. In addition, internal and externalelastic lamina and media disruptions at the injury site wereevident, often with media substitution by fibrotic tissue.Quantitative analysis performed 30 days after injury showed asignificantly 36% larger carotid lumen CSA with MSC treatmentin comparison to control DMEM-treated rats (P< 0.05)


(Fig. 6A). Also the carotid area encompassed by the EEL, anindex of arterial remodeling, resulted to be significantlydecreased in DMEM-treated rats in comparison toMSC-treated rats (Fig. 6B), indicating that MSC treatmentstrongly reduces the deleterious inward remodeling, a majordeterminant of luminal narrowing in restenosis. Finally, the CSAof the Mþ I significantly increased after injury both in MSC- andin DMEM-treated rat carotids in comparison to contralateraluninjured carotids, but no significant difference was detectablebetween the two groups of arteriotomy-injured carotids(Fig. 6C).

MSCs affect the expression of inflammation-relatedgenes and of the differentiation marker SM22

We extracted total RNA from injured and contralateraluninjured carotids harvested at 3 and 7 days after arteriotomyfrom MSC- (n¼ 4 for each time point) or DMEM-treated rats(n¼ 4 for each time point) and from carotids of uninjured rats(n¼ 3). We analyzed through RT-PCR the differentialexpression of the inflammation-related genes IL-1a, IL-1b, IL-6,IL-10, Mcp-1, TGF-b, TNF-a, and IFN-g. Expression dataobtained in injured were compared to expression levels incarotids from uninjured rats.

Mcp-1 mRNA was detectable only in arteriotomy-injuredcarotids and it showed a significant 1.5- and 1.82-fold decreasein MSC-treated rats in comparison to DMEM-treated rats at3 and 7 days after arteriotomy, respectively (P< 0.05) (Fig. 7A).Similarly, IL-1b mRNA was detectable only inarteriotomy-injured carotids both at 3 and 7 days afterarteriotomy, and it showed a significant threefold decrease inMSC-treated rats in comparison to DMEM-treated rats 7 daysafter injury (P< 0.05) (Fig. 7B).

Of note, basal levels of TGF-b mRNA observed in uninjuredcarotids did not change after arteriotomy in DMEM-treated ratcarotids, but it showed a significant 1.68- and 1.37-fold increaseonly in MSC-treated rats at 3 and 7 days after arteriotomy,respectively (P< 0.05) (Fig. 7C).

IL-6 and TNF-a mRNAs were detectable only inarteriotomy-injured carotids harvested 3 days afterarteriotomy, but without significant differences betweenMSC- and DMEM-treated rats.

We were unable to detect a signal for mRNAs coding forIL-1a, IL-10, and IFN-g both in uninjured and inarteriotomy-injured carotids.

Finally, we examined by RT-PCR the differential expressionof von Willebrandt Factor (vWF), a marker of ECs, and of SM22,a marker of differentiated SMCs. The mRNA coding for vWFshowed a progressive 4.3- and 6.6-fold decrease at 3 and 7 daysafter arteriotomy respectively in comparison to carotids fromuninjured rats, but without significant differences betweenMSC- and DMEM-treated carotids. SM22 mRNA also showed amarked 19- and 17-fold decrease at 3 and 7 days afterarteriotomy respectively, in comparison to carotids fromuninjured rats, but it revealed to be 2.7-fold higher inMSC-treated carotids in comparison to DMEM-treated rats at3 days after arteriotomy (P< 0.05) (Fig. 7D).

MSCs do not affect the expression ofinflammation-related genes in contralateraluninjured carotids

Our previous studies highlighted relevant changes of mRNAlevels not only in injured carotids but also in contralateraluninjured carotids, related to systemic inflammatory reactionand to vasocompensative reactions (Forte et al., 2008).Consequently, we investigated the effect of MSC administrationon mRNA expression also in contralateral uninjured carotids at3 and 7 days after arteriotomy. RT-PCR analysis revealed asignificant increase of inflammation-related factors Mcp-1,


TGF-b and IL-1b in comparison to carotids harvested fromuninjured rats, but no significant difference between MSC- andDMEM-treated rats (data not shown).

DiscussionBone marrow-derived rat MSCs retain multipotentpotentiality, high proliferative activity and lowsenescence before injection in vivo

The application of stem cells for therapeutic purposes requiresan in-depth knowledge of their biological characteristics, arigorous application of standardized methods and acharacterization of cells before administration in vivo.Multipotent MSCs promise to be a valuable therapeutic toolbut, due to their low number, require considerable in vitroexpansion before use in experimental models of disease and inclinical trials. This can lead to in vitro senescence andsubsequently to a decreased potential for proliferation anddifferentiation (Bonab et al., 2006). For these reasons, beforeMSC administration in rats submitted to arteriotomy, weverified their biological characteristics in order to set upsuitable culture conditions aimed at limiting MSC senescencewhile retaining their differentiation potential. Moreover, weapplied the minimal criteria for defining multipotent MSCs, assuggested by the International Society for Cellular Therapy(ISCT) (Dominici et al., 2006). In agreement with these criteria,we isolated MSCs on the basis of their adherence to plastics andwe verified that rat bone marrow-derived MSCs were able todifferentiate into bone and fat cells (Fig. 1B–D) and expressedthe surface markers CD73, CD90, and CD105 (Fig. 2B–D). Wealso verified that MSCs expressed SM-alpha actin (Fig. 2A),another feature of MSCs that provides them with contractileability (Ball et al., 2007).

MSCs are able to home at the injury site and to reducesurgically-induced inward remodeling of rat carotids

We successfully treated rats with allogenic MSCs without anyimmunosuppressive protocol, in agreement with previousstudies revealing their low immunogenicity (Patel et al., 2008),probably related to the lack on cell surface of the majorhistocompatibility complex II (MHC II), that is responsible forimmune rejection (Le Blanc et al., 2003). Some studies revealedthat a subset of MSCs is positive for MHC II but they equallyexerted a veto-like activity on immune response (Potian et al.,2003).

New evidence suggests that the artery wall is a recipient andsource of MSCs (Abedin et al., 2004; Hoshino et al., 2008). Ourdata indicate that DiI-labeled MSCs injected via tail vein in ratwere able to home in the carotid vascular wall since 3 days aftervascular injury, with a preferential localization in the adventitiaand in the perivascular tissue (Fig. 4F,G). Our previous report(Forte et al., 2007b) revealed the up-regulation of mRNAsinvolved in stem cell homing, and in particular a maximal4.73-fold increase of the chemokine Cxcl12 (or SDF-1a), ableto mediate the mobilization of bone marrow-derived SMCprogenitor cells through the corresponding receptor Cxcr4,that also increased. No DiI-labeled MSCs were detectable incontralateral uninjured carotids (Fig. 4A,E), neither an increaseof Cxcl12 and Cxcr4 mRNAs (Forte et al., 2008), thussuggesting that engraftment of MSCs requires tissue injury.

It is well known that cell trapping in the pulmonarymicrovasculature can significantly reduce the number of MSCsthat can access injured organs, as demonstrated by interestingstudies in mice (Schrepfer et al., 2007). These authorsdemonstrated that the vast majority of MSCs, whose diameteris about 15–19 mm, are trapped in lung capillaries withinseconds after intravenous injection, and that this phenomenoncan be reduced by pretreatment with sodium nitroprusside, asource of nitric oxide. Nevertheless, we demonstrated in our


study that the MSCs that were able to escape pulmonarytrapping were sufficient to limit the negative remodeling inarteriotomy-injured rat carotids, in agreement with otherstudies underlining that the number of MSCs necessary inregenerative medicine protocols do not necessarily need to behigh, as MSCs are presumed to play their reparative andanti-inflammatory action mainly through soluble factors, playinga paracrine action (Psaltis et al., 2008). Moreover, the rate ofpost-surgery death in MSC-treated rats was very low (about5%), indicating that injected MSCs did not cause relevantpathophysiological problems. Many other therapeuticprotocols in animal models and in patients based on intravenousadministration of MSCs revealed to be effective and safe,despite the possible cell trapping in the pulmonarymicrovasculature (Ma et al., 2005; Lee et al., 2008).

The morphological and morphometric data resulting fromthis study support the therapeutic potentiality of MSCs invascular (re)stenosis. In agreement with our previous studies(Forte et al., 2001), the morphological analysis of rat carotids at30 days after rat carotid arteriotomy highlighted relevantdifferences in comparison to carotids submitted to balloonangioplasty, since we observed a marked lumen stenosis mainlyto due neoadventitia and to negative or inward remodeling,while the role of intima hyperplasia proved quite limited(Fig. 5B,D). Inward remodeling is a phenomenon that involvesextracellular matrix and, in particular, collagen synthesis andspatial redistribution, as well as elastic fiber accumulation.Inward remodeling is currently considered to be the main causeof restenosis (Pasterkamp et al., 2000). The morphometricresults reveal a significant reduction of carotid inwardremodeling together with an increase of lumen CSA (Fig. 6A)but not of the Mþ I CSA in MSC-treated rats (Fig. 6C),indicating that the difference in inward remodeling betweenMSC- and DMEM-treated rats is most probably related toadventitial cicatrization, as previously demonstrated by porcineangioplasty studies (Zalewski and Shi, 1997), involving changesin matrix composition and conformation that can induce vesselshrinkage.

MSCs are able to modulate the expression ofinflammation-related genes and of SM22 marker atthe injury site in rat carotids

Three main hypotheses can be proposed to explain the positiverole played by MSCs in limitation of negative remodelinginduced by arteriotomy of rat carotid: (1) circulating MSCscould be recruited at the injury site by chemokines releasedafter arteriotomy and then differentiate to SMCs and/or to ECs,thus inducing a rapid regeneration of vascular tissue andconsequently limiting vascular cell proliferation. In this context,studies based on co-culture experiments revealed that a directcontact between MSCs and SMCs is required in thedifferentiation of MSCs into SMCs (Wang et al., 2006);(2) recruited MSCs could produce a variety of cytokines,including VEGF and bFGF, that could exert a paracrine action onresident vascular cells at the injury site and promote a rapidrecovery of damage; (3) MSCs could home at the injury site andexert a local and/or systemic immunosuppressive action thuslimiting the inflammatory reaction and other related processes,including reactive oxygen species production and cellproliferation and migration. These three hypotheses are notmutually exclusive but could coexist in this pathophysiologicalprocess. The microarray analysis of functional pathwaysrevealed a marked inflammatory reaction in arteriotomy-injured carotids (Forte et al., 2007b). On the basis of theimmunomodulatory properties of MSCs and of the key role thatinflammation plays in vascular stenosis, we aimed to gain insightin the mechanism of action of MSCs in vascular stenosis byverifying the differential expression of some of the mostsignificant markers of inflammation.

Fig. 5. MSCs are effective in reducing arteriotomy-induced stenosis. Representative cross-sections of carotids from MSC- and DMEM-treatedWKY rats, harvested 30 days after arteriotomy. Contralateral uninjured carotid from MSC-treated rat (A). Injured carotid fromMSC-treated rat (B). Contralateral uninjured carotid from DMEM-treated rat (C). Injured carotid from DMEM-treated rat (D). Red arrowsindicate the point in the adventitia where the suture polypropilene stitch was applied after arteriotomy. Hematoxylin-orcein staining,10T magnification.

796 F O R T E E T A L .

TGF-b is an immunosuppressive cytokine whose mRNAincreased at 3 and 7 days after injury only in MSC-treatedcarotids (Fig. 7C). The increased production of TGF-b by MSCshomed at the injury site could induce a suppression of the localimmune response, and in particular of T-lymphocyteproliferation (Di Nicola et al., 2002; Nasef et al., 2007). TGF-bhas pleiotropic effects on cardiovascular cells (Ruiz-Ortegaet al., 2007). In particular, it acts as a potentantiproliferative mediator on SMCs (Seay et al., 2005).Moreover, the increase of TGF-b mRNA in MSC-treatedcarotids in comparison to control DMEM-rats could have aneffect also on myofibroblast differentiation, as it is well knownthat TGF-b plays a major role in the wound healing process(Hinz, 2007).

IL-1b is a proinflammatory cytokine able to induce theexpression of cyclooxygenase 2 and type 2 phospholipase A,and also to act as a hematopoietic growth factor (Dinarello,2002). IL-1b expression is activated by vascular injury and thereduction of its mRNA at 7 days after injury and MSC treatment(Fig. 7B) could positively affect the local inflammatory reactionin injured carotids, in agreement with other studies (Guo et al.,2007; Koide et al., 2007).

Similarly, Mcp-1 is a proinflammatory chemokine able tostimulate SMC proliferation and macrophage infiltration ininjured vascular tissue (Schober and Zernecke, 2007). Mcp-1expression in carotids was activated by vascular injury; the


reduction of its mRNA at 3 and 7 days after MSC treatment(Fig. 7A) is in agreement with other observations (Ohnishi et al.,2007) and could positively affect the outcome of carotid surgicalinjury. This hypothesis is supported by previous studiesdemonstrating that Mcp-1 inhibition reduces restenosis indifferent models of injury (Furukawa et al., 1999; Schober et al.,2004).

On the basis of these RT-PCR preliminary results we canhypothesize that MSCs reduce carotid stenosis at least in partthrough a synergic inhibition of the inflammation bymacrophage invasion and proinflammatory cytokine expressionand through the activation of anti-inflammatory/antiproliferative pathways.

The increase of inflammation-related mRNAs we detectednot only in arteriotomy-injured rat carotids but also incontralateral uninjured carotids when compared to arteriesfrom uninjured rats is in agreement with our previousobservations (Forte et al., 2008) and can be related to asystemic inflammatory reaction triggered by carotidarteriotomy. The injection of MSCs in rats affects theexpression of inflammation-related genes (Mcp-1, IL-1b, andTGF-b) only at the injury site where they home, and not at distaluninjured vascular beds, thus revealing, at least in ourexperimental protocol, that they do not affect the immunereaction at systemic level. Further studies on serum factors willbe necessary to further analyze this aspect.

Fig. 6. Morphometric measurements on cross-sections ofMSC-treated rat carotids 30 days after the surgical injury. LumenCSA (A), EEL area (B), and I R M CSA (C) in injured and incontralateral uninjured carotids harvested 30 days after arteriotomyand MSC or DMEM treatment. Data are presented as themean W SEM. P < 0.05 versus DMEM-treated carotids wasconsidered statistically significant.

Fig. 7. MSCs affect the expression of mRNAs coding for moleculesinvolved inflammatory-immune reaction and for SM22 inarteriotomy-injured carotids. Mcp-1 (A), IL-1b (B), TGF-b (C) andSM22 (D) mRNA analysis by RT-PCR on total RNA extracted at 3 and7 days after arteriotomy and MSCs or DMEM treatment and fromcarotids from uninjured rats. All measurements were normalizedwith respect to endogenous GAPDH levels. The values are expressedin arbitrary units as relative changes over the normalized uninjuredcontrol. Data are presented as the mean W SEM. P < 0.05 versusDMEM-treated carotids was considered statistically significant.


SMCs within adult blood vessels retain remarkable plasticityand can undergo profound and reversible changes in phenotypein response to environmental influences, such as vascular injury(Owens et al., 2004). Characterization of contractile andproliferative SMCs has lead to the identification of a number ofdifferentiation markers under the control of the SerumResponse Factor, including SM22 (Shanahan and Weissberg,1998; Regan et al., 2000). The decrease of SM22 mRNA inarteriotomy-injured carotids in comparison to carotids fromuninjured rats is in agreement with our previous observations(Forte et al., 2005) and suggests an arteriotomy-induced shift ofSMCs from a contractile to a proliferative phenotype. MSCtreatment transiently limits the loss of SM22 expression(Fig. 7D), possibly contributing to a limitation of SMCproliferation.

The decrease of vWF we observed at 3 and 7 days after injuryis in agreement with previous microarray data (Forte et al.,2007b) and with a partial apoptosis-mediated loss of ECs afterarteriotomy revealed by TUNEL assay (Forte et al., 2005). TheMSC treatment did not contribute to limit the vWF mRNAdecrease, thus suggesting that the mechanism of action ofMSCs, at least in this model of vascular injury, does not involve arapid endothelial recovery.


Conclusions

MSCs are multipotent self-renewing cells that retain theflexibility to differentiate into various lineages. Owing tothese features, and to the ease of ex vivo expansion, there is ahuge expectation from the use of MSCs for therapeuticapplications.

This study represents a first step for the comprehension ofthe role of rat bone marrow-derived allogenic MSCs insurgically induced vascular stenosis. Our results indicate that asystemic intravenous administration of allogenic MSCs is safe,

798 F O R T E E T A L .

as we did not detect any evident adverse effect in MSC-treatedrats and the rate of post-surgery death was very low.

MSCs resulted to home at the injury site in a model of carotidarteriotomy and were able to reduce lumen stenosis. Themolecular events leading to tissue repair, including vascular wallrepair after arteriotomy, remain a mysterious and complexprocess. Our data indicate that MSCs are effective in reducingarteriotomy-induced inward remodeling, in contrast with otherstudies conducted in rats arteries submitted to angioplasty(Chen et al., 2007) and in mice arteries submitted to wire injury(Wang et al., 2008), respectively, thus demonstrating that theeffectiveness of a MSC-based therapy is strongly related to thekind of vascular injury to be repaired.

Among all the possible pathways that led to this positiveoutcome, we provided information about a localimmunomodulatory role for MSCs and their contribution tomaintain at least in part SMCs in a differentiated phenotype.

Several clinical trials with MSCs are already ongoing fordifferent diseases. They have been used for the first stem/progenitor cell-based clinical trials in patients with osteogenesisimperfecta (Horwitz et al., 2001) and then in patients withmucopolysaccharidosis (Koc et al., 2002). Subsequently, trialswere initiated for graft-versus-host disease, that are based onthe ability of the MSCs to suppress immune reactions. Ourfindings can have clinical implications for prevention orreduction of restenosis in patients submitted to vascular injuryinvolving the full wall thickness, such as carotidendarterectomy, arterial grafting or transplant vasculopathy.Nevertheless, a deeper understanding of the mechanisms ofaction of MSCs in (re)stenosis in other pre-clinical studies isrequired before trials in patients.

Acknowledgments

We are grateful to Ms. Maria Rosaria Cipollaro, Dr. TizianaSquillaro and Dr. Nicola Alessio for skilful assistance. SHRO2007–2008 ‘‘Role of cell cycle-related genes in the biology ofstem cells’’ grant to U.G.; Legge 5 Regione Campania 2003‘‘Modello sperimentale di iperplasia fibrointimale post-chirurgica in modelli di ratto affetti da patologie dell’apparatocardiovascolare’’ to M.D.F.; Progetto Finalizzato Sanita 2003‘‘Patologie infettive e insulto chirurgico: studi di genomica eproteomica nel remodeling vascolare’’ to A.C.

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Mesenchymal stem cells effectively reduce surgically induced stenosis in rat carotids