Coadministration of Endothelial and Smooth Muscle Progenitor Cells Enhances the Efficiency of Proangiogenic Cell-Based Therapy
Cell-based therapy is a promising approach designed to enhance neovascularization and function of ischemic tissues. Interaction between endothelial and smooth muscle cells regulates vessels development and remodeling and is required for the formation of a mature and functional vascular network. Therefore, we assessed whether coadministration of endothelial progenitor cells (EPCs) and smooth muscle progenitor cells (SMPCs) can increase the efficiency of cell therapy. Unilateral hindlimb ischemia was surgically induced in athymic nude mice treated with or without intravenous injection of EPCs (0.5×106), SMPCs (0.5×106) and EPCs+SMPCs (0.25×106+0.25×106). Vessel density and foot perfusion were increased in mice treated with EPCs+SMPCs compared to animals receiving EPCs alone or SMPCs alone (P<0.001). In addition, capillary and arteriolar densities were enhanced in EPC+SMPC–treated mice compared to SMPC and EPC groups (P<0.01). We next examined the role of Ang-1/Tie2 signaling in the beneficial effect of EPC and SMPC coadministration. Small interfering RNA directed against Ang-1–producing SMPCs or Tie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporated EPCs within vascular structure, and abrogated the efficiency of cell therapy. Production of Ang-1 by SMPCs activates Tie2-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 types of vascular progenitor cells may constitute a novel therapeutic strategy for improving the treatment of ischemic diseases.
Cell-based therapy is a promising strategy to induce neovessel formation in patients with peripheral artery diseases or myocardial ischemia.1 Administration of bone marrow–derived mononuclear cells (BM-MNCs) or endothelial progenitor cells (EPCs) has been shown to improve postischemic neovascularization in various experimental studies and clinical trials.2–6 Infused progenitor cells are recruited to ischemic tissues and may first contribute to neovascularization and tissue/vessel remodeling through paracrine effects.7–9 Another primary role of progenitor cells may be to incorporate into blood vessels and regenerate the vascular endothelial barrier; however, their incorporation rate is very low.10,11 Moreover, recent studies have shown that age and other risk factors for cardiovascular diseases reduce the availability of progenitor cells and impair their function to varying degrees, likely limiting the efficiency of autologous stem cell therapy.12–15 Therefore, strategies to improve the therapeutic potential of cell therapy need to be developed to counteract progenitor cells dysfunction in patients with cardiovascular risk factors.16–18
Although the neovascularization process involves different cell types and various growth factors, most of the cell therapy protocols are based on the biological effects of single cell type population, ie, EPCs or on administration of heterogeneous population of progenitors, ie, BM-MNCs or peripheral blood MNCs, characterized by a high scarcity of vascular progenitor cells. A tight cooperation between endothelial cells and smooth muscle cells/pericytes is critical for the development of functional neovessels. In addition, stabilization of neovessels regulates blood flow vascular permeability and also endothelial cell functions, such as proliferation, survival, and migration.19 Endothelial and smooth muscle cells cooperate to regulate vessel maturation and stability. In particular, angiopoietins (Angs) and their receptor Tie2 have been shown to participate to the communication between endothelial and mural cells.20 In addition, direct administration of plasmid encoding for Ang-1 increases postischemic revascularization after hindlimb ischemia and myocardial infarction.21,22 Moreover, the receptor tyrosine kinase Tie2 is expressed on endothelial cells and hematopoietic stem cells.23 Ang-1 interaction with its receptor Tie2 induces intracellular signaling promoting endothelial cells survival, migration, sprouting, and network formation.24
Therefore, one can hypothesize that coadministration of endothelial and smooth muscle progenitor cells may trigger the formation of more stable and functional vascular networks. In a previous work, we have isolated and differentiated EPCs and smooth muscle progenitor cells (SMPCs) from human umbilical cord blood.25 In the present study, we assessed whether coadministration of EPCs and SMPCs can increase the efficiency of cell therapy in a model of operatively induced hindlimb ischemia. We also analyzed the role of Ang-1/Tie2 signaling in EPC and SMPC cooperation.
Materials and Methods
EPCs and SMPCs were isolated from human umbilical cord blood and differentiated ex vivo as previously described.25
Mouse Model of Unilateral Hindlimb Ischemia
All the experiments were performed in accordance with the European Community guidelines for the care and use of laboratory animals (No. 07430). Eight-week-old male athymic Nude mice (Harlan, France) underwent surgical ligature of the proximal part of the right femoral artery, as previously described.26,27 Six hours after the onset of ischemia, PBS, EPCs (0.5×106), SMPCs (0.5×106) and EPCs plus SMPCs (0.25×106+0.25×106, respectively) were intravenously injected (10 mice per group). In some experiments, SMPCs were transfected with small interfering (si)RNA directed against Ang-1 and EPCs with siRNA directed against Tie2 before cell transplantation. After 2 weeks of treatment, vessel density was evaluated by high-definition microangiography, capillary density analysis, and laser Doppler perfusion imaging to assess in vivo tissue perfusion in the paw, as previously described.26,27
Ischemic and nonischemic gastrocnemius muscles were collected and progressively frozen in isopentane solution cooled in liquid nitrogen. Cross-sections (6 μm) were fixed in 100% cold acetone and incubated for 1 hour with a rat anti-mouse CD31 monoclonal antibody (clone MEC 13.3, BD Biosciences, Le Pont de Claix, France) or a rabbit anti-mouse α-smooth muscle actin (αSMA) polyclonal antibody (Laboratory Vision, Francheville, France). Vessels number were evaluated per muscle fiber and then expressed as a ratio of ischemic to nonischemic leg.
EPC Detection in Ischemic Muscles
To demonstrate incorporation of EPCs into ischemic muscles, EPCs (1×106 cells per 100 μL of PBS) were intravenously administered 6 hours after induction of hindlimb ischemia as described above. The gastrocnemius muscles were harvested 4 days after injection of EPCs. Frozen tissue sections (10 μm) were prepared and fixed with ice cold acetone. Incorporated EPCs were detected by immunostaining with a biotinylated antihuman CD31 antibody (DAKO ARK, DAKO) followed by incubation with streptavidin–Alexa 568. To confirm incorporation of human cells, mouse vasculature was stained with anti-mouse CD31 antibody. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Invitrogen). A total of 10 fields was evaluated with fluorescence microscopy to detect EPC incorporation.
siRNA Transfection Protocol
Ang-1– and Tie2-specific siRNA duplexes (siGENOME SMARTpool) and nontargeting control siRNA (Luciferase siRNA) were purchased from Dharmacon (Brebières, France). Briefly, cells were grown on 6-well plates until 80% confluence. The siRNA solution was mixed with serum-free and antibiotic-free medium M199 containing DharmaFECT2 siRNA Transfection reagent. The culture medium was removed and replaced with 800 μL of antibiotic-free M199 medium containing 8% FCS, and 200 μL of the transfection mix was added to each well to achieve a final siRNA concentration of 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 Media
Conditioned media were collected from SMPCs after 48 hours of transfection with siRNA directed against Ang-1 and then analyzed by ELISA kit for Ang-1 (R&D Systems, Lille, France).
EPC and SMPC Cocultures on Matrigel
Matrigel (BD Biosciences, Le Pont de Claix, France) was added into a 12-well plate. EPCs were labeled with SP-Dioc18 green dye (2 μg/mL) (Invitrogen), and SMPCs were labeled with CM-DiI red dye (1 μg/mL) (Invitrogen). EPCs were added to Matrigel (12×105 EPCs per well) and then incubated overnight in medium M199 containing 10% FCS. SMPCs were then added to the endothelial network (0.4×105 SMPCs per well) for 8 hours. Cells were visualized by fluorescence using inverted-phase microscope (Zeiss, Le Pecq, France). For quantitative analysis, SMPC coverage was determined as a ratio of the number of SMPCs to total area of endothelial cells network using HistoLab and Saisam software (Microvision Instruments, Paris, France).
EPC Tube Formation on Matrigel
EPCs were added (12×105 per well) and incubated overnight in medium M199 containing 10% FCS to induce tube formation. Conditioned media from Ang-1 siRNA-transfected SMPCs or control siRNA-transfected SMPCs were then added overnight at 37°C. In other experiments, EPCs transfected with Tie2 siRNA were incubated and cultured in SMPC conditioned media overnight at 37°C. For quantitative analysis, Matrigel wells were observed under an Axiovert 25 microscope (Zeiss), and Saisam software (Microvision Instruments) was used to count the number of sprouts in 10 fields of each well.
Incorporation of EPCs Into the Human Umbilical Vein Endothelial Cell Network Formation
EPCs (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 conditioned media. Cells were visualized by fluorescence using inverted-phase microscope. For quantitative analysis, the number of incorporated EPCs into the HUVEC network was determined as a ratio of the number of EPCs to total area of HUVEC network using HistoLab and Saisam software (Microvision Instruments).
Quantification of EPC Apoptosis
Tie2 siRNA-transfected EPCs were incubated with serum-free medium containing human recombinant Ang-1 (50 ng/mL) (R&D Systems, Lille, France) or with conditioned medium of SMPCs. The presence of apoptotic cells was evaluated by fluorescence-activated cell sorting (FACS) analysis using Annexin V and propidium iodide staining. After 24 hours, cells were washed twice with PBS and stained using the Apoptosis Detection kit I (BD Biosciences). Analysis was carried out using a FACSCalibur flow cytometer within 1 hour of staining. A total of 8000 events was analyzed using CELLQuest software (BD Biosciences). Annexin V–positive cells indicate cells undergoing apoptosis, whereas Annexin V– and propidium iodide–positive cells are scored as necrosis.
Protein lysates (20 μg) were separated by electrophoresis in 4% to 12% acrylamide gels containing sodium dodecyl sulfate and transferred to nitrocellulose membranes. Membranes were incubated for with goat antihuman Tie2 polyclonal antibody (R&D Systems) and then with horseradish peroxidase–conjugated anti-goat IgG (Jackson ImmunoResearch, Villepinte, France).
Results are expressed as means±SEM. One-way ANOVA was used to compare variables. Comparisons between 2 groups were performed using nonparametric Mann–Whitney test. A value of P<0.05 was considered significant.
Coadministration of EPCs and SMPCs Activates Postischemic Neovascularization
We first assessed the ability of EPC and SMPC coinjection to promote neovascularization. For this purpose, EPCs were intravenously coinjected with equal number of SMPCs into mice with femoral artery ligature. Two weeks later, angiographic score (vessel density), subcutaneous blood flow (foot perfusion), and capillary and arteriolar density were analyzed. Phenotypic characterization of EPCs and SMPCs is provided in Figure I in the online data supplement.
SMPC injection alone did not significantly increase the angiographic score compared to PBS receiving mice. Administration of EPCs increased by 65% vessel density in ischemic leg compared to PBS-treated mice (P<0.01) (Figure 1A), as shown previously.27 Interestingly, angiographic score was further increased by 75% in mice treated with both EPCs and SMPCs compared to those transplanted with EPCs alone (P<0.001).
Laser Doppler Perfusion Imaging
SMPC injection alone slightly enhanced foot perfusion in ischemic limb compared to PBS receiving mice. Intravenous administration of EPCs increased by 52% the ischemic/nonischemic foot blood perfusion ratio versus PBS-treated mice (P<0.001; Figure 1B). Coadministration of EPCs+SMPCs further raised by 66% paw perfusion in reference to EPC-treated mice (P<0.001).
Capillary and Arteriolar Density
EPC transplantation alone enhanced by 50% capillary density versus PBS-receiving mice (P<0.01). In EPC+SMPC–treated mice, the ratio of ischemic to nonischemic leg capillary density was increased by 116%, 97%, and 64% compared to PBS, SMPC, and EPC groups, respectively (Figure 2, left). Interestingly, immunohistological analysis of ischemic muscles sections stained with an anti-αSMA antibody also revealed an increase in the number of αSMA-positive cells in mice treated with EPCs+SMPCs (Figure 2, right). These results demonstrated that coadministration of EPCs and SMPCs enhanced capillary and arteriolar densities in the ischemic hindlimb.
Incorporation of EPCs Into Ischemic Muscles
We also evaluated the number of incorporated EPCs into the mouse microvasculature (green labeling) by fluorescent staining directed against human CD31 (red labeling) (Figure 3). Histological and quantitative analyses showed that the number of incorporated EPCs was significantly greater in the EPC+SMPC group compared to EPCs alone. It is noteworthy that we were unable to detect dioc 488–labeled SMPCs in our experimental conditions. To further demonstrate that SMPCs were not localized within the ischemic tissues, we evaluated human GAPDH mRNA levels in mouse ischemic hindlimb using RT-PCR. Human GAPDH was detected in ischemic limbs of mice treated with EPCs and EPCs+SMPCs but not with those treated with SMPCs alone. GAPDH levels were also higher in EPC+SMPC–treated mice compared to those treated with EPCs alone (175±17% versus 100±21%, respectively; P<0.05; n=6).
Beneficial Effect of EPC and SMPC Bitherapy: Role of Ang-1
We next examined the potential mechanisms by which EPC and SMPC bitherapy improved postischemic neovascularization. Interaction between endothelial cells and mural cells is a key feature in the regulation of vascular formation and stabilization.20 This cooperation is controlled by several systems such as the Ang-1/Tie2 signaling.20 As shown in Figure 4A, the release of Ang-1 was markedly increased in supernatants 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 siRNA markedly impaired the release of Ang-1 by SMPCs compared to SMPCs treated with control siRNA (400±15 pg/mL versus 1960±103 pg/mL respectively; P<0.001) (Figure 4A). Therefore, we analyzed the role of Ang-1–producing SMPCs on EPC-related functions.
First, we tested the role of Ang-1 in the coverage capacity of SMPCs on the EPC-induced endothelial cell networks, using a Matrigel coculture assay. SMPCs transfected with Ang-1 siRNA failed to cover the preformed endothelial cell networks 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 leakage, induce cell migration and tube formation, and prevent cell death.24 We therefore analyzed the role of Ang-1 released by SMPCs on EPC-induced capillary tube formation. Conditioned media from Ang-1 siRNA-transfected SMPCs hampered the preformed endothelial cell networks and reduced the number of sprouts compared to the conditioned media from control siRNA-transfected SMPCs (Figure 4C). Interestingly, addition of recombinant Ang-1 in conditioned media from Ang-1 siRNA-transfected SMPCs restores endothelial cell networks stabilization and sprouting (Figure 4C).
We next sought to define the role of SMPC-released Ang-1 in the beneficial effect of SMPC and EPC cotherapy. For this purpose, SMPCs were transfected with Ang-1 siRNA and then combined with EPCs before their transplantation in mice with hindlimb ischemia. Interestingly, inhibition of SMPC-released Ang-1 reversed the beneficial effect of EPC+SMPC coinjection during postischemic revascularization (Figure 5). This effect was associated with a reduction in the homing and incorporation of EPCs into the mouse vasculature (Figure 3). Finally, we were not able to detect human Ang-1 levels in blood of SMPCs and SMPC+EPC–treated animals. Altogether, these results suggest that SMPC administration does not induce a systemic upregulation of Ang-1 levels and that SMPCs modulate EPC-related effects through local and paracrine release of Ang-1.
Beneficial Effect of EPC and SMPC Bitherapy: Role of Tie-2
Ang-1 is a ligand for Tie2 receptor expressed on EPCs.27 We therefore evaluated the role of Tie-2 in the beneficial effect of EPC and SMPC coadministration. EPCs were transfected with siRNA directed against the Tie2 transcript. Western blot analysis indicated that Tie2 siRNA decreased by around 85% Tie2 expression in transfected EPCs compared to control siRNA (Figure 6A). In vitro capillary tube formation on Matrigel showed that knockdown of Tie2 expression in EPCs significantly reduced their ability to form networks in the presence of SMPC conditioned media and in presence of 50 ng/mL human recombinant Ang-1 (rhAng-1), underscoring the role of the Ang-1/Tie2 system in EPC-induced tube formation on Matrigel (Figure 6B).
To gain more insights into the role of Tie2 on EPC and SMPC interaction, we particularly focused our study on the SMPC capacity to cover the EPC-induced endothelial cell networks transfected with or without Tie2 siRNA. In Tie2 siRNA-transfected EPC networks, the coverage capacity of SMPCs was reduced compared to EPCs transfected with control 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 EPCs to incorporate into capillary-like structures. The number of incorporated CM DiI-labeled EPCs was decreased by transfection with Tie2 siRNA compared to control siRNA (0.33±0.09 versus 0.18±0.04, respectively; P<0.05) (Figure 7A), suggesting that Ang-1/Tie-2 signaling is involved in EPC incorporation into the vascular endothelium.
Ang-1/Tie2 signaling has been shown to mediate antiapoptotic activity in endothelial cells.24,28 Reduction of tube formation in Tie2 siRNA-transfected EPCs could be mediated 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 conditioned media from SMPCs. FACS analysis of Annexin V expression showed 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 Tie2 siRNA-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 therapeutic effect of coadministration of EPCs and SMPCs (Figure 8). In addition, siRNAs directed against Tie-2–expressing EPCs reduced the homing and incorporation of EPCs into the mouse vasculature compared to EPC+SMPC group (Figure 3).
Taken together, these results suggest that coadministration of EPCs and SMPCs increased postischemic neovascularization through a cell cooperation involving Ang-1/Tie2 system.
The main findings of this study were that (1) combined administration of EPC and SMPC derived from human umbilical cord blood was more efficient than EPC or SMPC cell transplantation alone and (2) this effect resulted from the release of Ang-1 by SMPCs and subsequent activation of Tie2-expressing EPCs.
The postischemic neovascularization process involves a coordinated interplay of different cell types mainly vascular and inflammatory cells and various growth factors that ensure the development of mature blood vessels.29 However, most of the proangiogenic cell therapy protocols are based on the injection of EPCs only or on heterogeneous populations of stem cells containing a low proportion of vascular progenitors, which is probably not sufficient to induce maturation and stabilization of neovessels. We provide evidence, for the first time, that cotransplantation of EPCs and SMPCs could be more appropriate to tightly orchestrate the complex process of neovascularization. In line with these findings, Matrigel implants containing EPCs derived from cord blood combined with human saphenous vein smooth muscle cells (hSVSMCs) revealed a significant increase in microvessel density compared to Matrigel implants containing EPCs or hSVSMCs alone.30 Combined transplantation of human embryonic stem cell–derived endothelial cells and mural cells also raised therapeutic neovascularization.31 Endothelial cell and smooth muscle cell cooperation is critical for the development of mature vascular networks.19 The intercellular communication between these 2 cell types is regulated, at least in part, by transforming growth factor-β, platelet-derived growth factor-BB, shingosine-1-phosphate, and Angs. In the present study, we showed that SMPC-derived from umbilical cord blood produced a large amount of Ang-1. Ang-1 released by SMPCs may first control SMPC-related effects. Indeed, we showed that SMPC-released Ang-1 can promote their ability to cover preformed endothelial cells networks in a Matrigel coculture assay. Ang-1 may also directly affect the preexistent vascular networks. In this view, Ang-1 has been reported to significantly increase arteriolar density in ischemic areas.22,32 Similarly, we showed that combined administration of EPCs and SMPCs upregulated microvascular density with increased number of αSMA-positive smooth muscle cells/pericytes. We also demonstrated that SMPC-released Ang-1 can facilitate stabilization of endothelial cell networks. Ang-1 has been shown to prevent endothelial cells from apoptosis and induce sprouting and migration,24 suggesting that SMPC-released Ang-1 may also control endothelial cell network formation.
Finally, Ang-1 may act on Tie2-expressing EPCs. The resulting intracellular signaling regulates endothelial cells survival and vascular remodeling.33 Disruption of Tie2 function led to early embryonic lethality, with defects in the microvasculature.34 Therefore, we examined the potential role of Tie2 receptor in the cell cooperation between Tie2-expressing EPCs and Ang-1–producing SMPCs. Interestingly, transfection of EPCs using Tie2 siRNA reduces their ability to form capillary-like networks on Matrigel and hampers their incorporation rate in HUVECs in presence of either SMPC conditioned media or rhAng-1. We demonstrated that these effects are likely mediated by an increase in EPC apoptosis. More interestingly, siRNAs directed against Ang-1–producing SMPCs or Tie2-expressing EPCs blocked vascular network formation in Matrigel coculture assays, reduced the rate of incorporated EPCs within the mouse vasculature, and abrogated the efficiency of cell therapy. In this view, administration of a plasmid encoding Ang-1 with autologous bone marrow cell injection enhances neovascularization in a rabbit hindlimb ischemia model compared with BM-MNC implantation alone.35 These results suggest that the increase efficiency of cotherapy results from the release of Ang-1 by SMPCs, leading to the activation of Tie2 at the EPC surface and subsequently to the activation of EPC-related functions. Nevertheless, it is likely that SMPCs have other effect than just a secretion of Ang-1. In this view, SMPC conditioned medium inhibited apoptosis in siRNA Tie2-transfected cells, suggesting that SMPC antiapoptotic effect was not mediated by Ang-1, only.
Proangiogenic cell therapy is an attractive option for the treatment of ischemic diseases. Clinical trials in patients with critical limb ischemia or myocardial infarction provide evidence that autologous cell therapy can improve the neovascularization process. Nevertheless, these protocols are currently limited by several parameters: a paucity of progenitor cells in the peripheral circulation and a low rate of incorporation of cells in ischemic areas. Moreover, progenitor cells functions are reduced in patients with cardiovascular risk factors (diabetes, hypertension, hypercholesterolemia, etc).18 Therefore, novel strategies to improve the efficiency of proangiogenic cell-based therapy need to be developed. Our study proposed a new concept that coadministration of EPCs and SMPCs may improve the therapeutic effect of cell therapy, especially in aged patients with cardiovascular risk factors.
In conclusion, our study demonstrates that combined administration of EPCs and SMPCs enhances postischemic revascularization through a cooperative action between 2 two types of progenitors that involves the Ang-1/Tie2 pathway. The present findings may open the way for the development of novel proangiogenic strategies based on the administration of SMPCs or other mural progenitors with BM-MNCs, peripheral blood MNCs, or EPCs to improve the efficiency of stem cell therapy in patients with peripheral artery occlusive and ischemic heart diseases.
We thank Téni Ebrahimian and Ludovic Waeckel (Institut National de la Santé et de la Recherche Médicale U689, Paris, France) for technical help. We also thank Patrice Castagnet (Laboratoire d’anatomopathologie at Lariboisière Hospital, Paris, France) for excellent assistance in tissue embedding and processing. We are grateful to the maternity participants of Lariboisière Hospital for providing us with cord blood samples.
Sources of Funding
This work was supported, in part, by grants from the Agence Nationale de la Recherche (Cardiovascular, Obesity and Diabetes, ANR-05-028-01 ANR-05-022-01 and ANR-05-022-01) and the Del Duca Fundation. J.-S.S. was supported by grants from the Agence Nationale de la Recherche (ANR-05-JCJC-0065-01) and Fondation de France.
Original received March 4, 2008; revision received July 31, 2008; accepted August 12, 2008.
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