Coadministration of Endothelial and Smooth Muscle Progenitor Cells Enhances the Efficiency of Proangiogenic Cell-Based Therapy

Coadministration of Endothelial and Smooth MuscleProgenitor Cells Enhances the Efficiency of Proangiogenic

Cell-Based TherapyPhilippe Foubert, Gianfranco Matrone, Boussad Souttou, Carole Lere-Dean, Veronique Barateau,

Jean Plouet, Sophie Le Ricousse-Roussanne, Bernard I. Levy, Jean-Sebastien Silvestre, Gerard Tobelem

Abstract—Cell-based therapy is a promising approach designed to enhance neovascularization and function of ischemictissues. Interaction between endothelial and smooth muscle cells regulates vessels development and remodeling and isrequired for the formation of a mature and functional vascular network. Therefore, we assessed whether coadminis-tration of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) can increase the efficiencyof cell therapy. Unilateral hindlimb ischemia was surgically induced in athymic nude mice treated with or withoutintravenous injection of EPCs (0.5�106), SMPCs (0.5�106) and EPCs�SMPCs (0.25�106�0.25�106). Vessel densityand foot perfusion were increased in mice treated with EPCs�SMPCs compared to animals receiving EPCs alone orSMPCs alone (P�0.001). In addition, capillary and arteriolar densities were enhanced in EPC�SMPC–treated micecompared to SMPC and EPC groups (P�0.01). We next examined the role of Ang-1/Tie2 signaling in the beneficialeffect of EPC and SMPC coadministration. Small interfering RNA directed against Ang-1–producing SMPCs orTie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporatedEPCs within vascular structure, and abrogated the efficiency of cell therapy. Production of Ang-1 by SMPCs activatesTie2-expressing EPCs, resulting in increase of EPC survival and formation of a stable vascular network. Subsequently,the efficiency of EPC- and SMPC-based cotherapy is markedly increased. Therefore, coadministration of different typesof vascular progenitor cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.(Circ Res. 2008;103:751-760.)

Key Words: angiogenesis � progenitor cells � ischemia � angiopoietin-1 � Tie2

Cell-based therapy is a promising strategy to induceneovessel formation in patients with peripheral artery

diseases or myocardial ischemia.1 Administration of bonemarrow–derived mononuclear cells (BM-MNCs) or endothe-lial progenitor cells (EPCs) has been shown to improvepostischemic neovascularization in various experimentalstudies and clinical trials.2–6 Infused progenitor cells arerecruited to ischemic tissues and may first contribute toneovascularization and tissue/vessel remodeling throughparacrine effects.7–9 Another primary role of progenitor cellsmay be to incorporate into blood vessels and regenerate thevascular endothelial barrier; however, their incorporation rateis very low.10,11 Moreover, recent studies have shown that ageand other risk factors for cardiovascular diseases reduce theavailability of progenitor cells and impair their function tovarying degrees, likely limiting the efficiency of autologousstem cell therapy.12–15 Therefore, strategies to improve thetherapeutic potential of cell therapy need to be developed tocounteract progenitor cells dysfunction in patients with car-diovascular risk factors.16–18

Although the neovascularization process involves differentcell types and various growth factors, most of the cell therapyprotocols are based on the biological effects of single celltype population, ie, EPCs or on administration of heteroge-neous population of progenitors, ie, BM-MNCs or peripheralblood MNCs, characterized by a high scarcity of vascularprogenitor cells. A tight cooperation between endothelialcells and smooth muscle cells/pericytes is critical for thedevelopment of functional neovessels. In addition, stabiliza-tion of neovessels regulates blood flow vascular permeabilityand also endothelial cell functions, such as proliferation,survival, and migration.19 Endothelial and smooth musclecells cooperate to regulate vessel maturation and stability. Inparticular, angiopoietins (Angs) and their receptor Tie2 havebeen shown to participate to the communication betweenendothelial and mural cells.20 In addition, direct administra-tion of plasmid encoding for Ang-1 increases postischemicrevascularization after hindlimb ischemia and myocardialinfarction.21,22 Moreover, the receptor tyrosine kinase Tie2 is

Original received March 4, 2008; revision received July 31, 2008; accepted August 12, 2008.From the Institut des Vaisseaux et du Sang (P.F., B.S., C.L.-D., J.P., S.L.R.-R., G.T.), Paris; and Cardiovascular Research Center (J.-S.S., G.M., V.B.,

J.P., B.I.L.), Institut National de la Sante et de la Recherche Medicale Lariboisiere U689, Universite Paris 7, Hopital Lariboisiere, Paris, France.Correspondence to Gerard Tobelem, Institut des Vaisseaux et du Sang, 8 rue Guy Patin, 75475 Paris Cedex 10, France. E-mail

[email protected]© 2008 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.108.175083

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expressed on endothelial cells and hematopoietic stem cells.23

Ang-1 interaction with its receptor Tie2 induces intracellularsignaling promoting endothelial cells survival, migration,sprouting, and network formation.24

Therefore, one can hypothesize that coadministration ofendothelial and smooth muscle progenitor cells may triggerthe formation of more stable and functional vascular net-works. In a previous work, we have isolated and differenti-ated EPCs and smooth muscle progenitor cells (SMPCs) fromhuman umbilical cord blood.25 In the present study, weassessed whether coadministration of EPCs and SMPCs canincrease the efficiency of cell therapy in a model of opera-tively induced hindlimb ischemia. We also analyzed the roleof Ang-1/Tie2 signaling in EPC and SMPC cooperation.

Materials and MethodsCell CultureEPCs and SMPCs were isolated from human umbilical cord bloodand differentiated ex vivo as previously described.25

Mouse Model of Unilateral Hindlimb IschemiaAll the experiments were performed in accordance with the Euro-pean Community guidelines for the care and use of laboratoryanimals (No. 07430). Eight-week-old male athymic Nude mice(Harlan, France) underwent surgical ligature of the proximal part ofthe right femoral artery, as previously described.26,27 Six hours afterthe onset of ischemia, PBS, EPCs (0.5�106), SMPCs (0.5�106) andEPCs plus SMPCs (0.25�106�0.25�106, respectively) were intra-venously injected (10 mice per group). In some experiments, SMPCswere transfected with small interfering (si)RNA directed againstAng-1 and EPCs with siRNA directed against Tie2 before celltransplantation. After 2 weeks of treatment, vessel density wasevaluated by high-definition microangiography, capillary densityanalysis, and laser Doppler perfusion imaging to assess in vivo tissueperfusion in the paw, as previously described.26,27

ImmunohistochemistryIschemic and nonischemic gastrocnemius muscles were collectedand progressively frozen in isopentane solution cooled in liquidnitrogen. Cross-sections (6 �m) were fixed in 100% cold acetoneand incubated for 1 hour with a rat anti-mouse CD31 monoclonalantibody (clone MEC 13.3, BD Biosciences, Le Pont de Claix,France) or a rabbit anti-mouse �-smooth muscle actin (�SMA)polyclonal antibody (Laboratory Vision, Francheville, France). Ves-sels number were evaluated per muscle fiber and then expressed asa ratio of ischemic to nonischemic leg.

EPC Detection in Ischemic MusclesTo demonstrate incorporation of EPCs into ischemic muscles, EPCs(1�106 cells per 100 �L of PBS) were intravenously administered 6hours after induction of hindlimb ischemia as described above. Thegastrocnemius muscles were harvested 4 days after injection ofEPCs. Frozen tissue sections (10 �m) were prepared and fixed withice cold acetone. Incorporated EPCs were detected by immunostain-ing with a biotinylated antihuman CD31 antibody (DAKO ARK,DAKO) followed by incubation with streptavidin–Alexa 568. Toconfirm incorporation of human cells, mouse vasculature was stainedwith anti-mouse CD31 antibody. Nuclei were stained with 4�,6-diamidino-2-phenylindole (DAPI) (Invitrogen). A total of 10 fields wasevaluated with fluorescence microscopy to detect EPC incorporation.

siRNA Transfection ProtocolAng-1– and Tie2-specific siRNA duplexes (siGENOME SMART-pool) and nontargeting control siRNA (Luciferase siRNA) werepurchased from Dharmacon (Brebieres, France). Briefly, cells weregrown on 6-well plates until 80% confluence. The siRNA solution

was mixed with serum-free and antibiotic-free medium M199containing DharmaFECT2 siRNA Transfection reagent. The culturemedium was removed and replaced with 800 �L of antibiotic-freeM199 medium containing 8% FCS, and 200 �L of the transfectionmix was added to each well to achieve a final siRNA concentrationof 50 nmol/L. Transfected cells were incubated at 37°C for 48 hours,and protein expression was analyzed by ELISA or western blotting.

Detection of Ang-1 in SMPC-Conditioned MediaConditioned media were collected from SMPCs after 48 hours oftransfection with siRNA directed against Ang-1 and then analyzedby ELISA kit for Ang-1 (R&D Systems, Lille, France).

EPC and SMPC Cocultures on MatrigelMatrigel (BD Biosciences, Le Pont de Claix, France) was added intoa 12-well plate. EPCs were labeled with SP-Dioc18 green dye (2�g/mL) (Invitrogen), and SMPCs were labeled with CM-DiI red dye(1 �g/mL) (Invitrogen). EPCs were added to Matrigel (12�105 EPCsper well) and then incubated overnight in medium M199 containing10% FCS. SMPCs were then added to the endothelial network(0.4�105 SMPCs per well) for 8 hours. Cells were visualized byfluorescence using inverted-phase microscope (Zeiss, Le Pecq,

Figure 1. Combination of EPCs and SMPCs increases neovas-cularization in hindlimb ischemia. EPCs (0.25�105) were intrave-nously injected with SMPCs (0.25�105) in ischemic mice.Shown are representative photomicrographs and quantitativeanalysis of angiographic score (A) and foot perfusion (B) evalu-ated 14 days after ischemia and cell transplantation. Valuesare expressed as means�SEM (n�10 per group). *P�0.05**P�0.01 vs PBS-injected mice; ##P�0.01, ###P�0.001 vsEPCs. ns indicates no significant statistical differences.

752 Circulation Research September 26, 2008

France). For quantitative analysis, SMPC coverage was determinedas a ratio of the number of SMPCs to total area of endothelial cellsnetwork using HistoLab and Saisam software (Microvision Instru-ments, Paris, France).

EPC Tube Formation on MatrigelEPCs were added (12�105 per well) and incubated overnight inmedium M199 containing 10% FCS to induce tube formation.Conditioned media from Ang-1 siRNA-transfected SMPCs or con-trol siRNA-transfected SMPCs were then added overnight at 37°C.In other experiments, EPCs transfected with Tie2 siRNA wereincubated and cultured in SMPC conditioned media overnight at37°C. For quantitative analysis, Matrigel wells were observed underan Axiovert 25 microscope (Zeiss), and Saisam software (Microvi-sion Instruments) was used to count the number of sprouts in 10fields of each well.

Incorporation of EPCs Into the Human UmbilicalVein Endothelial Cell Network FormationEPCs (3�104 per well) labeled with CM-DiI red dye (Invitrogen)were mixed with human umbilical endothelial cells (HUVECs)(12�105 per well) and then incubated overnight in SMPC condi-tioned media. Cells were visualized by fluorescence using inverted-phase microscope. For quantitative analysis, the number of incorpo-rated EPCs into the HUVEC network was determined as a ratio ofthe number of EPCs to total area of HUVEC network using HistoLaband Saisam software (Microvision Instruments).

Quantification of EPC ApoptosisTie2 siRNA-transfected EPCs were incubated with serum-free me-dium containing human recombinant Ang-1 (50 ng/mL) (R&DSystems, Lille, France) or with conditioned medium of SMPCs. Thepresence of apoptotic cells was evaluated by fluorescence-activatedcell sorting (FACS) analysis using Annexin V and propidium iodidestaining. After 24 hours, cells were washed twice with PBS andstained using the Apoptosis Detection kit I (BD Biosciences).Analysis was carried out using a FACSCalibur flow cytometerwithin 1 hour of staining. A total of 8000 events was analyzed usingCELLQuest software (BD Biosciences). Annexin V–positive cellsindicate cells undergoing apoptosis, whereas Annexin V– andpropidium iodide–positive cells are scored as necrosis.

Western BlottingProtein lysates (20 �g) were separated by electrophoresis in 4% to12% acrylamide gels containing sodium dodecyl sulfate and trans-ferred to nitrocellulose membranes. Membranes were incubated forwith goat antihuman Tie2 polyclonal antibody (R&D Systems) andthen with horseradish peroxidase–conjugated anti-goat IgG (JacksonImmunoResearch, Villepinte, France).

Statistical AnalysisResults are expressed as means�SEM. One-way ANOVA was usedto compare variables. Comparisons between 2 groups were per-formed using nonparametric Mann–Whitney test. A value of P�0.05was considered significant.

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Figure 2. Combination of EPCs and SMPCs increases the capillary and arteriolar densities. Shown are representative photomicro-graphs and quantitative analysis of histological sections in ischemic skeletal muscles. Ischemic muscles were costained for CD31(endothelial marker) and �SMA (smooth muscle cell marker). Arrows indicate �SMA-positive arterioles. Values are expressed asmeans�SEM (n�10 per group). **P�0.01 vs PBS-injected mice; ##P�0.01, ###P�0.001 vs EPCs. ns indicates no significant statisticaldifferences.

Foubert et al EPCs and SMPCs for Therapeutic Angiogenesis 753

ResultsCoadministration of EPCs and SMPCs ActivatesPostischemic NeovascularizationWe first assessed the ability of EPC and SMPC coinjection topromote neovascularization. For this purpose, EPCs wereintravenously coinjected with equal number of SMPCs intomice with femoral artery ligature. Two weeks later, angio-graphic score (vessel density), subcutaneous blood flow (footperfusion), and capillary and arteriolar density were analyzed.Phenotypic characterization of EPCs and SMPCs is providedin Figure I in the online data supplement.

Microangiography AnalysisSMPC injection alone did not significantly increase theangiographic score compared to PBS receiving mice. Admin-istration of EPCs increased by 65% vessel density in ischemicleg compared to PBS-treated mice (P�0.01) (Figure 1A), asshown previously.27 Interestingly, angiographic score wasfurther increased by 75% in mice treated with both EPCs andSMPCs compared to those transplanted with EPCs alone(P�0.001).

Laser Doppler Perfusion ImagingSMPC injection alone slightly enhanced foot perfusion inischemic limb compared to PBS receiving mice. Intravenousadministration of EPCs increased by 52% the ischemic/nonis-chemic foot blood perfusion ratio versus PBS-treated mice(P�0.001; Figure 1B). Coadministration of EPCs�SMPCsfurther raised by 66% paw perfusion in reference to EPC-treated mice (P�0.001).

Capillary and Arteriolar DensityEPC transplantation alone enhanced by 50% capillary densityversus PBS-receiving mice (P�0.01). In EPC�SMPC–treated mice, the ratio of ischemic to nonischemic legcapillary density was increased by 116%, 97%, and 64%compared to PBS, SMPC, and EPC groups, respectively(Figure 2, left). Interestingly, immunohistological analysis ofischemic muscles sections stained with an anti-�SMA anti-body also revealed an increase in the number of �SMA-positive cells in mice treated with EPCs�SMPCs (Figure 2,right). These results demonstrated that coadministration ofEPCs and SMPCs enhanced capillary and arteriolar densitiesin the ischemic hindlimb.

Incorporation of EPCs Into Ischemic MusclesWe also evaluated the number of incorporated EPCs into themouse microvasculature (green labeling) by fluorescent stain-ing directed against human CD31 (red labeling) (Figure 3).Histological and quantitative analyses showed that the num-ber of incorporated EPCs was significantly greater in theEPC�SMPC group compared to EPCs alone. It is noteworthythat we were unable to detect dioc 488–labeled SMPCs in ourexperimental conditions. To further demonstrate that SMPCswere not localized within the ischemic tissues, we evaluatedhuman GAPDH mRNA levels in mouse ischemic hindlimbusing RT-PCR. Human GAPDH was detected in ischemiclimbs of mice treated with EPCs and EPCs�SMPCs but notwith those treated with SMPCs alone. GAPDH levels were

also higher in EPC�SMPC–treated mice compared to thosetreated with EPCs alone (175�17% versus 100�21%, re-spectively; P�0.05; n�6).

Beneficial Effect of EPC and SMPC Bitherapy:Role of Ang-1We next examined the potential mechanisms by which EPCand SMPC bitherapy improved postischemic neovasculariza-tion. Interaction between endothelial cells and mural cells isa key feature in the regulation of vascular formation andstabilization.20 This cooperation is controlled by severalsystems such as the Ang-1/Tie2 signaling.20 As shown inFigure 4A, the release of Ang-1 was markedly increased insupernatants of cultured SMPCs as compared to that of EPCs(1850�20 pg/mL versus 226�14 pg/mL respectively;P�0.001). To investigate the role of SMPC-released Ang-1,we targeted Ang-1 with specific siRNA. Ang-1 siRNAmarkedly impaired the release of Ang-1 by SMPCs comparedto SMPCs treated with control siRNA (400�15 pg/mL versus1960�103 pg/mL respectively; P�0.001) (Figure 4A).

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Figure 3. Homing and incorporation of EPCs into ischemic mus-cles. Shown are representative photomicrographs and quantita-tive analysis of incorporated EPCs in histological sections fromischemic skeletal muscles. EPCs were stained using a biotinyl-ated antihuman CD31 antibody (red fluorescence). Mouse vas-culature was identified by CD31 staining (green fluorescence).Nuclei were stained with DAPI (blue fluorescence). Arrows indi-cate incorporated EPCs (n�5 per group). *P�0.05 vs EPC-injected mice; #P�0.05 vs EPC-injected mice. EPC indicatesmice treated with EPCs; SMPC, mice treated with SMPCs;EPC�SMPC siAng-1, mice treated with EPCs and SMPCstransfected with siRNA directed against Ang-1; EPC siTie-2�SMPC, mice treated with EPCs transfected with siRNAdirected against Tie-2 and SMPCs.


Therefore, we analyzed the role of Ang-1–producing SMPCson EPC-related functions.

First, we tested the role of Ang-1 in the coverage capacityof SMPCs on the EPC-induced endothelial cell networks,using a Matrigel coculture assay. SMPCs transfected withAng-1 siRNA failed to cover the preformed endothelial cellnetworks compared to SMPCs treated with control siRNA(0.3�0.016 versus 0.61�0.035, respectively; P�0.01)(Figure 4B).

Second, Ang-1 has been shown to suppress plasma leak-age, induce cell migration and tube formation, and preventcell death.24 We therefore analyzed the role of Ang-1 releasedby SMPCs on EPC-induced capillary tube formation. Condi-tioned media from Ang-1 siRNA-transfected SMPCs ham-pered the preformed endothelial cell networks and reducedthe number of sprouts compared to the conditioned mediafrom control siRNA-transfected SMPCs (Figure 4C). Inter-estingly, addition of recombinant Ang-1 in conditioned mediafrom Ang-1 siRNA-transfected SMPCs restores endothelialcell networks stabilization and sprouting (Figure 4C).

We next sought to define the role of SMPC-released Ang-1in the beneficial effect of SMPC and EPC cotherapy. For thispurpose, SMPCs were transfected with Ang-1 siRNA andthen combined with EPCs before their transplantation in micewith hindlimb ischemia. Interestingly, inhibition of SMPC-released Ang-1 reversed the beneficial effect of EPC�SMPCcoinjection during postischemic revascularization (Figure 5).This effect was associated with a reduction in the homing andincorporation of EPCs into the mouse vasculature (Figure 3).Finally, we were not able to detect human Ang-1 levels inblood of SMPCs and SMPC�EPC–treated animals. Alto-

gether, these results suggest that SMPC administration doesnot induce a systemic upregulation of Ang-1 levels and thatSMPCs modulate EPC-related effects through local andparacrine release of Ang-1.

Beneficial Effect of EPC and SMPC Bitherapy:Role of Tie-2Ang-1 is a ligand for Tie2 receptor expressed on EPCs.27 Wetherefore evaluated the role of Tie-2 in the beneficial effect ofEPC and SMPC coadministration. EPCs were transfectedwith siRNA directed against the Tie2 transcript. Western blotanalysis indicated that Tie2 siRNA decreased by around 85%Tie2 expression in transfected EPCs compared to controlsiRNA (Figure 6A). In vitro capillary tube formation onMatrigel showed that knockdown of Tie2 expression in EPCssignificantly reduced their ability to form networks in thepresence of SMPC conditioned media and in presence of 50ng/mL human recombinant Ang-1 (rhAng-1), underscoringthe role of the Ang-1/Tie2 system in EPC-induced tubeformation on Matrigel (Figure 6B).

To gain more insights into the role of Tie2 on EPC andSMPC interaction, we particularly focused our study on theSMPC capacity to cover the EPC-induced endothelial cellnetworks transfected with or without Tie2 siRNA. In Tie2siRNA-transfected EPC networks, the coverage capacity ofSMPCs was reduced compared to EPCs transfected withcontrol siRNA (0.48�0.05 versus 0.29�0.043, respectively;P�0.05) (Figure 6C).

We also analyzed the effect of Tie2 on the ability of EPCsto incorporate into capillary-like structures. The number ofincorporated CM DiI-labeled EPCs was decreased by trans-

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Figure 4. Role of Ang-1 in EPC and SMPC coopera-tion. A, ELISA measurement of Ang-1 protein levels inconditioned media of SMPCs transfected with Ang-1siRNA. B, Interaction of Ang-1 siRNA-transfected DiI-SMPCs (red) with EPC network (green) in Matrigelassay. Shown are representative photomicrographsand quantitative evaluation of DiI-SMPCs incorporatedinto EPC network. Arrows indicate incorporated DiI-SMPCs. C, Effect of SMPC-derived conditioned media(CM) in EPC-induced vascular network formation.Shown are representative photomicrographs andquantitative evaluation (number of sprouts) of EPC-induced vascular tube formation incubated overnightwith or without CM derived from Ang-1 siRNA-transfected SMPCs (n�3). ***P�0.001, **P�0.01,*P�0.05 vs SMPCs transfected with control siRNA. NTindicates nontransfected cells; Ctrl, control siRNA;CM, conditioned media.


fection with Tie2 siRNA compared to control siRNA(0.33�0.09 versus 0.18�0.04, respectively; P�0.05) (Figure7A), suggesting that Ang-1/Tie-2 signaling is involved inEPC incorporation into the vascular endothelium.

Ang-1/Tie2 signaling has been shown to mediate antiapo-ptotic activity in endothelial cells.24,28 Reduction of tubeformation in Tie2 siRNA-transfected EPCs could be medi-ated by an increase in EPC apoptosis. To test this hypothesis,Tie2 siRNA-transfected EPCs were incubated in serum-free

medium containing rhAng-1 (50 ng/mL) or in conditionedmedia from SMPCs. FACS analysis of Annexin V expressionshowed that whereas recombinant human Ang-1 (50 ng/mL)protected control siRNA-transfected EPCs from apoptosis(20%�4.4% annexin-V� cells), it did not protect Tie2siRNA-transfected EPCs (36.8%�2.5% annexin-V� cells)(Figure 7B). More interestingly, in hindlimb ischemia model,inhibition of Tie2 expression in EPCs reversed the therapeu-tic effect of coadministration of EPCs and SMPCs (Figure 8).In addition, siRNAs directed against Tie-2–expressing EPCsreduced the homing and incorporation of EPCs into the mousevasculature compared to EPC�SMPC group (Figure 3).

Taken together, these results suggest that coadministra-tion of EPCs and SMPCs increased postischemic neovas-cularization through a cell cooperation involving Ang-1/Tie2 system.

DiscussionThe main findings of this study were that (1) combinedadministration of EPC and SMPC derived from humanumbilical cord blood was more efficient than EPC or SMPCcell transplantation alone and (2) this effect resulted from therelease of Ang-1 by SMPCs and subsequent activation ofTie2-expressing EPCs.

The postischemic neovascularization process involves acoordinated interplay of different cell types mainly vascularand inflammatory cells and various growth factors that ensurethe development of mature blood vessels.29 However, most ofthe proangiogenic cell therapy protocols are based on theinjection of EPCs only or on heterogeneous populations ofstem cells containing a low proportion of vascular progeni-tors, which is probably not sufficient to induce maturationand stabilization of neovessels. We provide evidence, for thefirst time, that cotransplantation of EPCs and SMPCs couldbe more appropriate to tightly orchestrate the complex pro-cess of neovascularization. In line with these findings, Ma-trigel implants containing EPCs derived from cord bloodcombined with human saphenous vein smooth muscle cells(hSVSMCs) revealed a significant increase in microvesseldensity compared to Matrigel implants containing EPCs orhSVSMCs alone.30 Combined transplantation of human em-bryonic stem cell–derived endothelial cells and mural cellsalso raised therapeutic neovascularization.31 Endothelial celland smooth muscle cell cooperation is critical for the devel-opment of mature vascular networks.19 The intercellularcommunication between these 2 cell types is regulated, atleast in part, by transforming growth factor-�, platelet-derived growth factor-BB, shingosine-1-phosphate, andAngs. In the present study, we showed that SMPC-derivedfrom umbilical cord blood produced a large amount of Ang-1.Ang-1 released by SMPCs may first control SMPC-relatedeffects. Indeed, we showed that SMPC-released Ang-1 canpromote their ability to cover preformed endothelial cellsnetworks in a Matrigel coculture assay. Ang-1 may alsodirectly affect the preexistent vascular networks. In this view,Ang-1 has been reported to significantly increase arteriolardensity in ischemic areas.22,32 Similarly, we showed thatcombined administration of EPCs and SMPCs upregulated

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Figure 5. Treatment of SMPCs with Ang-1 siRNA reducesEPC�SMPC–induced postischemic neovascularization. SMPCswere transfected with Ang-1 siRNA and then combined withEPCs before intravenous injection. Angiographic score, foot per-fusion, and capillary and arteriolar densities were evaluated 14days later. Values are expressed as means�SEM (n�8 pergroup). **P�0.01, **P�0.001 compared with EPCs�SMPCstransfected with control (Ctrl) siRNA.


microvascular density with increased number of �SMA-positive smooth muscle cells/pericytes. We also demonstratedthat SMPC-released Ang-1 can facilitate stabilization ofendothelial cell networks. Ang-1 has been shown to preventendothelial cells from apoptosis and induce sprouting andmigration,24 suggesting that SMPC-released Ang-1 may alsocontrol endothelial cell network formation.

Finally, Ang-1 may act on Tie2-expressing EPCs. Theresulting intracellular signaling regulates endothelial cellssurvival and vascular remodeling.33 Disruption of Tie2 func-tion led to early embryonic lethality, with defects in themicrovasculature.34 Therefore, we examined the potentialrole of Tie2 receptor in the cell cooperation between Tie2-expressing EPCs and Ang-1–producing SMPCs. Interest-ingly, transfection of EPCs using Tie2 siRNA reduces their

ability to form capillary-like networks on Matrigel andhampers their incorporation rate in HUVECs in presence ofeither SMPC conditioned media or rhAng-1. We demon-strated that these effects are likely mediated by an increase inEPC apoptosis. More interestingly, siRNAs directed againstAng-1–producing SMPCs or Tie2-expressing EPCs blockedvascular network formation in Matrigel coculture assays,reduced the rate of incorporated EPCs within the mousevasculature, and abrogated the efficiency of cell therapy. Inthis view, administration of a plasmid encoding Ang-1 withautologous bone marrow cell injection enhances neovascu-larization in a rabbit hindlimb ischemia model compared withBM-MNC implantation alone.35 These results suggest that theincrease efficiency of cotherapy results from the release ofAng-1 by SMPCs, leading to the activation of Tie2 at the EPC

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Figure 6. Knockdown of Tie2 protein in EPCs reduces capillary tubeformation and SMPC coverage capacity. A, Tie2 knockdown inEPCs. Western blot quantification of Tie2 protein demonstrating theefficiency of Tie2 siRNA is shown. The membrane was reprobed foractin to check for equal loading. B, Capillary tube formation onMatrigel of EPCs treated with control or Tie2 siRNA and incubatedovernight with either 10% FCS or SMPC conditioned media (CM) orrhAng-1 (50 ng/mL). C, Representative photomicrographs and quanti-tative analysis of SMPC (red) coverage of control or Tie2 siRNA-transfected EPC networks (green) in Matrigel coculture assay. Arrowsindicate incorporated DiI-SMPCs. n�3, ***P�0.001, *P�0.05 vs con-trol (Ctrl) siRNA.


surface and subsequently to the activation of EPC-relatedfunctions. Nevertheless, it is likely that SMPCs have othereffect than just a secretion of Ang-1. In this view, SMPCconditioned medium inhibited apoptosis in siRNA Tie2-transfected cells, suggesting that SMPC antiapoptotic effectwas not mediated by Ang-1, only.

Proangiogenic cell therapy is an attractive option for thetreatment of ischemic diseases. Clinical trials in patients withcritical limb ischemia or myocardial infarction provide evi-dence that autologous cell therapy can improve the neovas-cularization process. Nevertheless, these protocols are cur-rently limited by several parameters: a paucity of progenitorcells in the peripheral circulation and a low rate of incorpo-ration of cells in ischemic areas. Moreover, progenitor cellsfunctions are reduced in patients with cardiovascular risk

factors (diabetes, hypertension, hypercholesterolemia, etc).18

Therefore, novel strategies to improve the efficiency ofproangiogenic cell-based therapy need to be developed. Ourstudy proposed a new concept that coadministration of EPCsand SMPCs may improve the therapeutic effect of celltherapy, especially in aged patients with cardiovascular riskfactors.

In conclusion, our study demonstrates that combined ad-ministration of EPCs and SMPCs enhances postischemicrevascularization through a cooperative action between 2 twotypes of progenitors that involves the Ang-1/Tie2 pathway.The present findings may open the way for the developmentof novel proangiogenic strategies based on the administrationof SMPCs or other mural progenitors with BM-MNCs,peripheral blood MNCs, or EPCs to improve the efficiency of

Figure 7. Knockdown of Tie2 protein in EPCs reduces their incorporation into the HUVEC network and their apoptosis. A, Representa-tive photomicrographs and quantitative analysis of incorporation DiI-labeled EPCs transfected with control or Tie2 siRNA into theHUVEC network in the presence of SMPC conditioned media (CM). B, Annexin V evaluation of EPC apoptosis by flow cytometry. Con-trol and Tie2 siRNA-transfected EPCs were incubated for 24 hours in serum-free medium containing rhAng-1 (50 ng/mL). The percent-age of apoptotic cells were characterized as those that stained with Annexin V and excluded propidium iodide (n�3). *P�0.05 vs con-trol (Ctrl) siRNA.


stem cell therapy in patients with peripheral artery occlusiveand ischemic heart diseases.

AcknowledgmentsWe thank Teni Ebrahimian and Ludovic Waeckel (Institut Nationalde la Sante et de la Recherche Medicale U689, Paris, France) fortechnical help. We also thank Patrice Castagnet (Laboratoired’anatomopathologie at Lariboisiere Hospital, Paris, France) forexcellent assistance in tissue embedding and processing. We are

grateful to the maternity participants of Lariboisiere Hospital forproviding us with cord blood samples.

Sources of FundingThis work was supported, in part, by grants from the AgenceNationale de la Recherche (Cardiovascular, Obesity and Diabetes,ANR-05-028-01 ANR-05-022-01 and ANR-05-022-01) and the DelDuca Fundation. J.-S.S. was supported by grants from the AgenceNationale de la Recherche (ANR-05-JCJC-0065-01) and Fondationde France.

DisclosuresNone.

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Figure 8. Treatment of EPCs with Tie2 siRNA (siTie2) reducesEPC�SMPC–induced postischemic neovascularization. EPCswere transfected with Tie2 siRNA and then combined withSMPCs before intravenous injection. Angiographic score, footperfusion, and capillary and arteriolar densities were evaluated14 days later. Values are expressed as means�SEM (n�8 pergroup). **P�0.01 compared with EPCs transfected with controlsiRNA (siCtrl). ##P�0.01, ###P�0.001 compared with EPCstransfected with control siRNA plus SMPCs.


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Coadministration of Endothelial and Smooth Muscle Progenitor Cells Enhances the Efficiency of Proangiogenic Cell-Based Therapy

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