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Cardiovascular Research 2004 62(1):176-184; doi:10.1016/j.cardiores.2004.01.017
© 2004 by European Society of Cardiology
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Copyright © 2004, European Society of Cardiology

Ex vivo differentiated endothelial and smooth muscle cells from human cord blood progenitors home to the angiogenic tumor vasculature

Sophie Le Ricousse-Roussanne*,a, Véronique Barateaua, Jean-olivier Contreresa, Bernadette Bovalb, Laurence Kraus-Berthierc and Gérard Tobelema

aInstitut des Vaisseaux et du Sang, Centre de Recherche de l'association Claude Bernard, Hôpital Lariboisière, 8 rue Guy Patin, 75475 Paris Cedex 10, France
bLaboratoire d'hématologie, Hôpital Lariboisière, 2 rue Ambroise Paré, 75475 Paris Cedex 10, France
cDivision of Experimental Cancerology, Institut de Recherche Servier, 92150 Suresnes, France

* Corresponding author. Tel.: +33-1-45262198; fax: +33-1-42829473. Email address: sophie.lericousse{at}lrb.ap-hop-paris.fr

Received 6 October 2003; revised 18 December 2003; accepted 8 January 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Recent studies have provided increasing evidence that postnatal neovascularization does not rely exclusively on sprouting of preexisting vessels, but also involves bone marrow-derived circulating endothelial precursors (BM-EPCs). Animal studies revealed that neovascularization of ischemic tissue can be enhanced by BM-EPCs transplantation. But a possible limitation to the use of vascular precursors for therapeutic angiogenesis is the relatively low number of these cells. In this study, we demonstrate that ex vivo expanded differentiated endothelial cells (ECs) and smooth muscle cells (SMCs), may home to the tumor vasculature allowing targeting of transgene expression to the neoangiogenic site. Methods: Mononuclear cells (MNCs) or CD34+-enriched cells were purified from cord blood. We have defined culture conditions in which we observed two types of clones easily differentiated according to their morphology: cobblestone or spindle-shaped. Phenotypic characterization was assessed by immunocytochemistry, flow cytometry analysis and polymerase reaction with reverse transcription. Formation of capillary-like network in vitro was studied in three-dimensional collagen culture. And recruitment of these cells to a tumoral neoangiogenic site was assessed into tumor-bearing Severe Combined Immunodeficient (SCID) mouse model. Results: The cobblestone cells uniformly positive for CD31, VE-cadherin, vWF, VEGF R1 and R2, ecNOS and incorporating acetylated LDL were ECs. Spindle-shaped cells expressed {alpha}-smooth muscle actin ({alpha}-SMA), Smooth Muscle Heavy Chain (SMHC), SM22 and calponin. They also displayed a carbachol-induced contractility in a medium containing IGF1. So we concluded that spindle-shaped cells were SMCs. ECs and SMCs interacted with each other to form a capillary like network in three-dimensional type I collagen culture. Moreover, these ex vivo differentiated cells are able to home to the tumor vasculature. Conclusion: We provide evidence that progenitors for ECs and SMCs circulate in human cord blood and differentiate into functional ECs and SMCs. These differentiated cells could provide a biomaterial for vascular cell therapy, because of their homing capacity to the neovascularization site.

KEYWORDS Progenitors; Vasculogenesis; Cord blood


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Growth of new blood vessels in the adult occurs through vasculogenesis, angiogenesis and arteriogenesis. ECs can initiate but not complete angiogenesis, periendothelial cells being essential for vascular maturation [1]. This maturation requires communication between ECs and supporting cells such as smooth muscle cells (SMCs) and pericytes [2–4]. In fact, once the primitive EC tubes are formed, the endothelium secretes factors that lead to the recruitment and/or induction of primordial smooth muscle cells, a process termed vascular myogenesis.

ECs arise from precursors called angioblasts or hemangioblasts in the embryo [5] or from circulating progenitors in the adult [6–10]. SMCs have a more complex origin, depending on their location. During embryogenesis, their precursors arise from three different lineages (mesenchymal cells, neural crest cells and epicardial-derived cells) [11]. SMCs could also transdifferentiate from ECs [12,13]. Moreover, several authors have postulated that ECs could give rise to a population of subendothelial cells during atherosclerotic intimal thickening [14,15]. Recent data suggest that SMCs may also originate from bone marrow-derived cells in the adult [16–18]. And the presence of smooth muscle progenitor cells has recently been shown in human blood [19]. Yamashita et al. [20] demonstrated that ECs and SMCs can also be derived from a common progenitor from embryonic stem cells, providing another pathway for vascular cell differentiation. Furthermore, it has been shown that freshly isolated human cord blood CD34+ cells injected into ischemic adductor muscles gave rise to endothelial and to skeletal muscle cells in mice [21]. Here we confirm that both progenitors of ECs and SMCs are present in cord blood and that they can differentiate into matured ECs and SMCs expressing specific markers and exhibiting specific functions. In vitro these two cell types collaborate with each other in three-dimensional culture to form a vascular-like structure and in vivo they are able to home to a neoangiogenic site without significant effect on total vessel density and tumor size. The fact that these cells retain their function after ex vivo expansion suggests that they could be used to target neovascularization sites in anti or pro-angiogenic therapies.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Purification of mononuclear cells and isolation of CD34+
Human umbilical cord blood samples (30–50 ml each) were collected in a sterile tube containing heparin sodium solution as anticoagulant from donors in compliance with French legislation. Mononuclear cells were isolated from cord blood by density gradient centrifugation with Pancoll (1.077 g/ml) (Dominique Dutscher, Brumath, France). The mononuclear cells were depleted of adherent cells by culturing on plastic dishes at 37 °C for 24 h and were directly plated into wells of six-well plates coated with type I collagen (Sigma-Aldrich, Saint Quentin Fallavier, France) or after a further cell subpopulation enrichment procedure (CD34+ cells). We isolated CD34+ cells from nonadherent cells using standard immunomagnetic techniques (CD34 isolation Kit, MACS; Miltenyi Biotech, Paris, France). Flow cytometry analysis of purified cells using a different clone (8G12) of FITC-conjugated anti-CD34 mAb showed that 75.0% (±5.6%) of the selected cells were positive for CD34. Isolated CD34+ cells (1.5 to 3.5 x 106 cells) were plated into a well of a six-well plate coated with a defined matrix containing fibronectin, laminin, heparan sulfate sodium, type I and type IV collagen (all purchased from Sigma-Aldrich) and hVEGF (R&D Systems, Oxford, UK).

2.2. Cell culture
Mononuclear cells (MNCs), purified CD34+ cells and Human Umbilical Vein Endothelial Cell (HUVEC) were cultured in M199 medium (Life Technologies, Cergy Pontoise, France) supplemented with 20% FCS, 15 mM HEPES, antibiotic and antimycotic solution (Life Technologies) and additional growth factors: hVEGF alone or with hIGF1 and hFGFb (all purchased from R&D Systems). Normal Human Dermal Fibroblast (NHDF) and Human Dermal Microvascular Endothelial Cell (HDMEC) (PromoCell, Heidelberg, Germany) were cultured in the medium given by the company. ECV 304 and EA hy 926 cell lines were cultured in M199 and RPMI 1640 (Life Technologies), respectively, supplemented with 10% FCS, 10 mM HEPES, 4 mM glutamin (Life Technologies) and antibiotic and antimycotic solution. Culture medium was changed twice a week. The human lung tumor cell line NCI-H460 (ATCC, Manassas, VA) was cultured in RPMI 1640 (Life technologies) supplemented with 10% heat inactivated fetal bovine serum, 2 mM L-glutamine, 100 units/ml penicillin, 100 mg/ml streptomycin, and 10 mM HEPES buffer pH 7,4. Ligand-induced contractility of cultured SMCs was analyzed as described [22].

2.3. Mice and tumor model
Female Severe Combined Immunodeficient (SCID) mice (age, 6–8 weeks; mean body weight, 18–20 g) were obtained from Iffa Credo (Lyon, France). The mice were housed in sterilized filter-topped cages and maintained in sterile conditions. All experiments involving animals were conducted in accordance with the Guide for the Care and Use of laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). Human NCI-H460 tumor cells (8 x 106 cells in a volume of 100 µl) were implanted subcutaneously in SCID mice previously treated i.p. with cyclophosphamide (150 mg/kg; day –2, day –1) on day 0. Three days after implantation, just before injection, the cells were labeled with PKH2-GL according to the manufacturers instructions (Sigma-Aldrich) and mice were injected via tail vein with 0.5 x 106 ECs or SMCs isolated from progenitors collected from cord blood and differentiated in culture conditions as described previously. Animals with tumors implanted but injected with PBS instead of human cells served as control. Nine days later, the animals were sacrificed, and tumor, spleen and liver were removed and fixed in 10% phosphate-buffered formalin and embedded in paraffin or immediately frozen in isopentane in liquid N2 for later inclusion in OCT compound. Sections of 5 µm were examined under a fluorescent microscope to visualize the incorporation of the fluorescent-labeled SMCs or ECs into the capillary networks.

2.4. Three-dimensional culture
Two milliliters of type I collagen at 1 mg/ml (Becton Dickinson) was dropped onto 35 mm dishes (Nunc, Fisher Scientific, Elancourt, France) and allowed to polymerize for 1 h at 37 °C. ECs, SMCs or fibroblasts were then seeded on top of the gel at high density alone or together in equal number for 24 h in culture medium with 10 ng/ml VEGF. Just before seeding, SMCs were labeled with PKH2-GL and cocultured with unlabeled ECs or fibroblasts. Formation of angiogenesis-like networks and incorporation of the fluorescent-labeled SMCs into the EC networks was examined under a fluorescent microscope.

2.5. Immunocytochemistry
Cells were grown on chamber slides (Lab-Tech, Poly Labo, Strasbourg, France) and fixed with cold 90% acetone solution. Nonspecific Ab binding was blocked by incubation of 10% normal goat serum (DAKO, Trappe, France) or FCS depending on the antibody used subsequently. We used as primary antibodies: polyclonal rabbit anti-human vWF (DAKO), a monoclonal mouse anti-human smooth muscle actin (1A4, DAKO), a polyclonal rabbit anti NOS3 (ecNOS) (C-20; sc654, Santa Cruz), and a monoclonal anti VEGF receptor 2 biotin conjugated (KDR, Sigma). We used ABC kit alkaline phosphatase goat IgG (Vector Laboratories, Compiègne, France) to detect smooth muscle actin label, a F(ab')2 fragment goat anti rabbit IgG (H+L) FITC (Coulter, Margency, France) to reveal vWF label, the EnVisionTM+ System Peroxidase (DAB) kit (DAKO) to detect NOS3 and the DAKO ARKTM kit (DAKO) to reveal VEGF R2. To identify capillaries, frozen sections from tumors were incubated with rabbit polyclonal antibody directed against total fibronectin (CHEMICON). Capillaries were demonstrated with a fluorescent TRITC anti-rabbit antibody (Coulter).

2.6. Cell lysis and Western immunoblotting for VEGF-R1
Cells were washed with ice-cold PBS and lysed in 1 ml of ice-cold lysis buffer containing 50 mM Tris pH8, 100 mM NaCl, 1% (v/v) Triton X-100, 0.5% (v/v) NP40, 50 mM EDTA, 1 mM PMSF, 10 µM leupeptin, 0.2 TIU/ml aprotinin, 2 mM sodium orthovanadate, 40 mM β-glycerophosphate, 50 mM NaF and 100 µM phenylarsine oxide. The cells were solubilized for 60 min at 4 °C. The homogenates were then centrifugated at 12,000 x g for 15 min. The supernatants were mixed with concentrated (4 x) Laemmli sample buffer, boiled for 5 min and centrifugated at 12,000 x g for 15 min. Protein lysates, corresponding to 4.5 x 105 cells, were separated by electrophoresis in 7.5% acrylamide/bisacrylamide (29:1) gels containing SDS and were transferred to nitrocellulose membranes in 25 mM Tris, 19 mM glycine and 15% methanol. Membranes were blocked by incubation in TBS containing 5% non-fat milk powder for 2 h at room temperature. The membranes were incubated overnight at 4 °C with antibody against VEGF-R1 (FLT-19, SIGMA) (1:200) in TBS, 0.1% Tween-20. The membranes were washed several times in TBS, 0.1% Tween-20, and were then incubated with horseradish peroxidase-conjugated anti-mouse IgG (1:5,000) in TBS, 0.1% Tween-20 for 2 h at room temperature. Antibody binding was detected with the Pierce enhanced chemiluminescence system (ECL), as recommended by the manufacturer.

2.7. Cellular uptake of acetylated LDL
Cells cultured on type I collagen were incubated in medium containing 10 µg/ml Dil-labeled Ac-LDL (TEBU, Le Perray en Yvelines, France) for 4 h at 37 °C. They were then, after fixation, examined under a fluorescence microscope.

2.8. Flow cytometry
An aliquot of cells was directly stained with Ab directed to CD31 (5.6E, Coulter), CD45/CD14 (2D1, M{varphi}P9, Becton Dickinson), CD51 (AMF7, Coulter), CD64 (22, Coulter), CD34 (8G12, Miltenyi Biotech), and CD133 (AC133/2, Miltenyi Biotech). We used as primary antibody F4-35H7 (Biocytex, Marseille, France) to stain CD146, and M3558 (DAKO) to stain Smooth Muscle Heavy Chain (SMHC) and a F(ab')2 goat anti-mouse IgG (H+L) FITC as secondary antibody (Coulter). Cells were stained with antibody anti-VE-cadherin (TEA1/31, Coulter) after a permeabilization step with IntraPrepTM permeabilization Reagent (Coulter). After staining, cells were fixed with 1% paraformaldehyde and analyzed by flow cytometry (FACStar flow cytometer, Becton Dickinson). FACS analyses were done on eight independent cobblestone clones (five isolated from monuclear cells and three isolated from CD34+ cells) and six independent spindle-shaped clones (three isolated from monuclear cells and three isolated from CD34+ cells). We did not observe any modification of any of these markers during the culture. To validate the specificity of the antibodies, we used different cells: NHDF, HDMEC, HUVEC, ECV 304 and EA hy 926 cell lines.

2.9. Reverse transcription-polymerase chain reaction (RT-PCR)
Total RNA was prepared with RNAXELR reagent (EUROBIO, Les Ulis, France) according to manufacturer's instructions. Both cDNA synthesis and PCR were performed in the same tube with SUPERSCRIPTTM One-Step RT-PCR (Life Technologies). We used the following primers: GAPDH forward: 5'-CCATGGAGAAGGCTGGGG-3', reverse: 5'-CAAAGTTGTCATGGATGACC-3', calponin forward: 5'-AGAAGTATGACCACCAGC-3', reverse: 5'-TAGAGCCCAATGATGTTCCG-3', SM22{alpha} forward: 5'-GCAGTCCAAAATTGAGAAGA-3', reverse: 5'-CTGTTGCTGCCCATTTGAAG-3', CD31 forward: 5'-AAGGTCAGCAGCATCGTGG-3', reverse: 5'-AGTGCAGATATACGTCCC-3', EphB4 forward: 5'-GAGAGGTACCTCCTGCAGTGTC-3', reverse: 5'-CCATGTCCGATGAGATACTGTCCG-3', ephrin B2 forward: 5'-CTGTGCCAGACCAGACCAAGA-3', reverse: 5'-CAGCAGAACTTGCATCTTGTC-3', VEGF-R2 forward: 5'-AACAAAGTCGGGAGAGGA-3', reverse: 5'-TGACAAGAAGTAGCCAGAAGA-3' and VEGF-R1 forward: 5'-CGACCTTGGTTGTGGCTGACT-3', reverse: 5'-CCCTTCTGGTTGGTGGCTTTG-3'.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Purification of progenitors of endothelial and smooth muscle cells
In order to investigate the presence of vascular progenitors in human cord blood, we cultured either MNCs on type I collagen in a medium containing 10 ng/ml VEGF or CD34+-enriched cells plated into six-well plates coated with a defined matrix in a medium containing 10 ng/ml VEGF, 1 ng/ml FGFb and 2 ng/ml IGF1. In both culture conditions we observed isolated starting clones after 7 to 15 days of culture. Surprisingly, these clones, which apparently grew randomly, were of two cell types easily differentiated according to their morphology, one type being cobblestone and the other having a spindle-shaped morphology (Fig. 1A,B). Colonies were picked and expanded for further analysis. These two cell types had a very high proliferative capacity up to 6 months for the cobblestone cells (35 passages) with a population doubling time of 24 h and up to 3 months for the spindle-shaped cells (15 passages) with a doubling time of 30 h.


Figure 1
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  		Fig. 1 Characterization of cobblestone and spindle-shaped cells. Outgrowth cell morphology at week 4 (A, B). Fluorescent immunostaining for vWB and acetylated-LDL uptake were studied for each type of cells. Cobblestone cells were positive for endothelial constitutive NO synthase (ecNOS). Cells were stained with an IgG control Ab or Ab against ecNOS followed by a peroxydase-coupled secondary Ab. Spindle-shaped cells were positive for smooth muscle actin ({alpha}-SMA) as demonstrated by immunostaining analysis. Flow cytometry analysis demonstrated that these cells also expressed SMHC. Flow cytometry analysis of cobblestone and spindle-shaped cells (C). In each graph, the dotted line outlines the region of fluorescent intensity for cells labeled with negative control antibody. The black line outlines the region identifying cells labeled with antibody for the expression marker indicated in each graph. These graphs were representative of eight independent cobblestone clones (c) (upper) and six independent spindle-shaped clones (s) (lower).

 
3.2. Cobblestone cells are ECs and spindle-shaped cells are SMCs
In order to determine the phenotype of both cell populations, we performed immunocytochemistry and flow cytometry analysis. The cobblestone population was uniformly positive for endothelial markers such as vWF (Fig. 1), endothelial constitutive NO synthase (ecNOS) (Fig. 1) and CD31 (86.0±15.2%), VE-cadherin (35% to 98% depending on the confluence of the culture), CD146 (S-endo1) (92.5±8.1%) (Fig. 1C) and negative for monocyte markers CD14, leucocyte marker CD45 and granulocyte marker CD64 (data not shown). In addition, these cells incorporated acetylated LDL (Fig. 1). Analysis by polymerase reaction with reverse transcription (RT-PCR) demonstrated that these cells expressed VEGF-R1 and R2 receptors (Fig. 2A). These results were confirmed by immunocytochemistry analysis for VEGF-R2 and Western blotting analysis for VEGF-R1 (Fig. 2B). This combined expression of specific endothelial markers and function unequivocally confirmed the identity of these cobblestone cells as an endothelial lineage.


Figure 2
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  		Fig. 2 RT-PCR analysis of ECs and SMCs markers in cobblestone cells (c), spindle-shape cells (s) and HDMECs (A). This last cell line was used as positive control for ECs. To confirm RT-PCR analysis, cobblestone cells were labeled with VEGF-R2 antibody (B). Negative control were performed with an IgG control Ab. Western blotting analysis confirmed that cobblestone cells expressed the VEGF-R1 (B).

 
In contrast, the spindle-shaped population was different according to these markers. In fact, they did not incorporate acetylated LDL and did not express vWF (Fig. 1), CD31, VE-cadherin (Fig. 1C). However, both types of cells expressed a high level of CD51 (cobblestone cells: 92.5±8.1% and spindle-shaped cells: 54.7±30.2%, Fig. 1C), but they had lost expression of CD34 (Fig. 1C) and CD133 (data not shown).

Immunocytochemistry and flow cytometry analysis demonstrated that the spindle-shaped cells expressed {alpha} smooth muscle actin (Fig. 1) and Smooth Muscle Heavy Chain (SMHC) (53,8±5,6%) (Fig. 1), specific markers for SMC [12]. RT-PCR confirmed that other SMC markers such as SM22{alpha} and calponin [23] were expressed in these cells (Fig. 2A). The spindle-shaped cells also displayed a carbachol-induced contractility in a medium containing 2 ng/ml IGF1 [22] (Fig. 3A,B). In contrast, these cells cultured under PDGF-BB-stimulated conditions were unable to contract (Fig. 3C,D). Taking into account these criteria, namely cell morphology, biochemical markers, and ligand-induced contractility, we concluded that the spindle-shaped cells were SMCs. It was interesting to note that ECs expressed EphB4, while this expression was very low in SMCs (Fig. 4). We also observed a very slight expression of ephrinB2 by the ECs (Fig. 4).


Figure 3
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  		Fig. 3 SMCs displayed a carbachol-induced contractility. The SMCs cultured on collagen I were stimulated with 2 ng/ml IGF1 (A, B) or 10 ng/ml PDGF-BB (C, D) for 3 days, and contraction was then induced by addition of 1 mM carbachol for 1 min. Photographs are shown before (A, C) and after (B, D) carbachol treatment. Arrow indicates loss of contact between two cells.

 

Figure 4
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  		Fig. 4 Expression of EphB4 receptor and ephrinB2 ligand by RT-PCR in ECs and SMCs.

 
3.3. ECs and SMCs interacted with each other to form capillary-like network in vitro
We next tested whether these two cell types collaborate with each other to form a vascular-like structure in vitro. In type three-dimensional collagen culture, we observed that ECs and SMCs migrate rapidly (within a few hours) to each other and interact to form a capillary-like network (Fig. 5C,F). When ECs were grown with the SMCs, they associated with each other and SMCs covered the tubular structure formed by ECs in only 24 h (Fig. 5C,F). ECs or SMCs cocultured with fibroblasts did not form any capillary-like network (data not shown). So these two cell types are functional and collaborate with each other.


Figure 5
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  		Fig. 5 ECs and SMCs collaborate with each other to form capillary-like network in vitro. ECs and SMCs were seeded on the top of the type I collagen gel and allowed to incubate for 24 h in a medium containing 10 ng/ml VEGF. Phase contrast microscopy (A–C). Fluorescent-labeled SMCs in the ECs network were examined under fluorescent microscope (D–F). When ECs were grown with SMCs, they associated with each other and SMCs covered the capillary formed by ECs (F).

 
3.4. ECs and SMCs were recruited to a tumoral neoangiogenic site
Recently, several studies reported that tumor angiogenesis is associated with recruitment of hematopoietic and circulating endothelial precursor cells [24–26]. To determine whether ex vivo differentiated SMCs and ECs home to the tumor vasculature, human lung tumor cell line NCI-H460 was implanted subcutaneously into SCID mice, and 3 days later PKH2-labeled ECs or SMCs were injected i.v. at a dose of 0.5 x 106 cells/mouse. At day 9 after tumor inoculation, the mice were sacrificed. No fluorescent cells were detected in tumors from mice injected with PBS (Fig. 6A). And, while no fluorescent cells were detected in liver or spleen, we observed homing of PKH2-labeled ECs and SMCs to the tumor vasculature. The ECs seemed to line the luminal surface of the vessels (Fig. 6B) while SMCs appeared to be located in the vascular wall (Fig. 6C). Incorporation of ECs or SMCs into the tumor vasculature may affect tumor angiogenesis, and thereby accelerate tumor vascularisation and tumor growth. To test this possibility, we analyzed the capillary density by fibronectin staining and the area of the tumor. In our protocol, no significant differences in capillary density (Fig. 7A) and the tumor weight (Fig. 7B,C) and were observed between control and cell transplanted tumors among the different groups.


Figure 6
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  		Fig. 6 Capacity of ECs and SMCs to be mobilized and incorporated into the tumor vasculature. Labeled progenitors derived ECs and SMCs (0.5 x 106 cells) were injected intravenously into SCID mice 3 days after implantation of a human lung tumor cell line (n=10). Nine days later, animals were sacrificed and tumors were removed, sectioned and analyzed by fluorescence microscopy. Green fluorescent signals indicate localization of transplanted human cells. No fluorescent cells were detected in tumors from mice injected with PBS (A). But fluorescent cells lining the luminal surface of the vessel were detected in animals perfused with ex vivo differentiated ECs (B). In mice perfused with ex vivo differentiated SMCs, fluorescent cells appeared to be located in the vascular wall (C).

 

Figure 7
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  		Fig. 7 Transplantation of ex vivo differentiated ECs or SMCs did not affect the total vessel density and tumor size. Ex vivo differentiated cells were injected via tail veins 3 days after human NCI-H460 non-small cell lung carcinoma cell-implantation was performed. Vascularized tumor was obtained 9 days after ex vivo differentiated cell implantation (A). No significant differences in tumor growth could be found between tumors from mice infused with human cells and tumors from mice injected with PBS (B). To identify capillaries, frozen sections from tumors were incubated with antibody directed against total fibronectin (C). There is no difference on total vessel density between animals injected with PBS or with ex vivo differentiated cells.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
In order to isolate the human vascular progenitors we purified either MNCs or CD34+ cells from human cord blood. Whatever the origin of the cells and the culture conditions, after 7 to 15 days of culture we observed isolated starting clones of two cell types easily differentiated according to their morphology. Taking into account their morphology, biochemical markers and functional capacity, we demonstrated that cobblestone cells were ECs and spindle-shaped cells were SMCs. For the spindle-shaped cells, simultaneous expression of multiple smooth muscle-specific proteins ({alpha}-smooth muscle actin, SM22, calponin and SMHC) is indicative of a smooth muscle phenotype. These differentiation markers relate to the contraction capacity of the cells [23] confirmed by the positive carbachol-induced contractility.

In our culture conditions, we observed a high expression of EphB4 on ECs, whatever the clone analyzed. Cells with venous fate express EphB4, the receptor for ephrin B2 [27]. Zhong et al. [28] demonstrated that angioblasts make up a limited pool of precursors whose default fate is venous, and the expression of gridlock further suppresses the venous characteristics and helps to drive the precursors towards formation of an aorta. It seems therefore that in our culture conditions, endothelial precursors retain their venous phenotype throughout their maturation process. The very slight expression of ephrin B2 in ECs could result from a high proliferative capacity. In fact, expression of ephrin B2 occurred in newly formed rather than preexisting vessels [29].

After 1 month of culture, both ECs and SMCs lost expression of CD34 and CD133, markers known to identify a population of functional endothelial precursors. However, this data does not presage the presence of mature ECs and SMCs in the starting cells. First, we depleted the starting cells of adherent cells by culturing them on plastic dishes for 24 h. In fact, several studies demonstrated no significant adherence of functional endothelial precursors within the first days of culture. Furthermore, both ECs and SMCs differentiated in vitro have a very high proliferative capacity as compared to adult mature vascular cells that exhibit less proliferative ability [9]. So isolated vascular cells from cord blood are progenitors able to differentiate within a few weeks of culture into matured ECs and SMCs. This is in line with the results of Simper et al. who recently demonstrated ex vivo outgrowth culture of SMCs from putative smooth muscle progenitor cells in human blood [19]. With CD34+-enriched cells, a single type of clones, spindle-shaped or cobblestone, was observed in a given well, each of them appearing with the same probability. This relatively high frequency of spindle-shaped cells from the purified CD34+ cells could result from our culture conditions (defined matrix and added growth factors). In contrast, with MNCs, ECs clones were more numerous than SMCs. In order to get more SMCs, we added in the culture medium PDGF-BB, a key factor for both SMC migration and proliferation. Contrary to the results of Yamashita et al. [16] and Simper et al. [19], we did not facilitate the SMCs differentiation in the presence of PDGF-BB (data not shown). In our culture conditions, PDGF-BB is, therefore, probably not necessary or not sufficient to commit more precursors to SMC lineage differentiation while VEGF was required. In agreement with our data, Hellström et al. [30] reported that formation of vascular SMC/pericytes occurs independently of PDGF-BB and PDGFR-β.

Recent data would support the assumption that the mesenchymal cells, often observed in primary EC cultures, are not the result of contamination but are often the result of transdifferentiation [31,32]. But this process seems to depend upon the presence of transforming growth factor-β1 and cell–cell contact. In our culture conditions, one cell type did not seem to come from a transdifferentiation process of the other one. We did not observe any "transitional" cells, coexpressing both endothelial and smooth muscle markers and once committed to a cell type, they did not exhibit phenotype modification whatever the growth factors added and the duration of culture.

The transplantation of human MNCs or CD34+ cells, expanded and committed to an endothelial lineage, was done in an animal ischemic model [24–26]. These experiments showed that ex vivo differentiated cells are incorporated into foci of neovascularization. In the case of tumor, it has been reported that CD34+ [32] from human cord blood or MNCs [33,34] from human blood may contribute to neoangiogenesis. Here we confirmed that ex vivo differentiated EPCs can participate in tumor neoangiogenesis after intravenous injection in SCID mouse that had received human NCI H460 tumor cells. And we showed, for the first time, that ex vivo differentiated and expanded SMCs can home to tumor vasculature. In our experimental conditions, analysis of tumor growth showed that incorporation of ECs or SMCs did not have a significant effect on total vessel density and tumor size. Our results are in line with other findings showing that ex vivo expanded mouse embryonic EPCs (eEPCs) can contribute to tumor-induced blood vessel growth in the adult without any differences in tumor growth and in total vessel density between animals injected with PBS or with eEPCs [35]. In contrary, De Bont et al. [25] demonstrated that i.v. infused human CD34+ mobilized cells enhance tumor growth in a xenotransplantation mouse NOD/SCID model of human non-Hodgkin's lymphoma. Tumor weight in the xenotransplanted mice infused with 0.5 x 106 human CD34+ cell is nearly twofold greater than that seen in xenotransplanted mice infused with control PBS. Moreover, they demonstrated a relation between the number of injected CD34+ cells and the increased tumor growth, suggesting that a certain number of injected CD34+ cells are needed to increase the size of the tumors. Even if we injected the same number of cells, ex vivo differentiated cells did not increase the tumor growth, suggesting that these cells might be different from CD34+ cells. A diminished recruitment or a step less effective in the multistep process during tumor angiogenesis described by Vajkoczy et al. [36] of ex vivo differentiated cells might be responsible for this observed phenomenon.

Due to the limited number of EPCs in the circulating blood, ex vivo expansion of EPCs appears necessary. Moreover, Yurugi-Kobayashi et al. demonstrated that differentiated vascular progenitor cells (VPC) from ES cells injected subcutaneously into tumor-bearing mice contribute to neovascularisation differently according to the differentiation stage [35]. Whereas undifferentiated VPC were often detected as non-vascular cells, differentiated VPC were more specifically incorporated into developing vasculature. We developed differentiation and expansion conditions that should preserve functions of ECs and SMCs especially homing to a neoangiogenic site, allowing targeting of transgene expression to a tumor neovasculature. Moreover, as demonstrated by Berger et al. [37], it could be a benefit to target both pericytes and ECs in the tumor vasculature for anti-angiogenic therapies. ECs and SMCs, differentiated ex vivo, could target the different constituents of the tumor vasculature, consequently rendering anti-angiogenic therapies more broadly efficacious.


   Acknowledgements
 
We thank the maternity hospital of Hôpital Lariboisière, Paris, France and of Hôpital Jean Rostand, Ivry sur Seine, France for cord blood samples.


   Notes
 
{star} Supported by grants from la Ligue de la Recherche contre le Cancer and l'Association pour la Recherche sur le Cancer.

Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

  1. Carmeliet P. Mechanisms of angiogenesis and arteriogenesis. Nat. Med. (2000) 6:389–395.[CrossRef][Web of Science][Medline]
  2. Hanahan D. Signaling vascular morphogenesis and maintenance. Science (1997) 277:48–50.[Free Full Text]
  3. Yancopoulo G.D., Davis S., Gale W.N., et al. Vascular-specific growth factors and blood vessel formation. Nature (2000) 407:242–248.[CrossRef][Medline]
  4. Folkman J., D'Amore P.A. Blood vessel formation: what is its molecular basis? Cell (1996) 87:1153–1155.[CrossRef][Web of Science][Medline]
  5. Choi K., Kennedy M., Kazarov A., Papadimitriou J.C., Keller G.A. Common precursor for hematopoietic and endothelial cells. Development (1998) 125:725–732.[Abstract]
  6. Asahara T., Murohara T., Sullivan A., et al. Isolation of putative progenitor endothelial cells for angiogenesis. Science (1997) 275:964–967.[Abstract/Free Full Text]
  7. Shi B.Q., Rafii S., Wu M.H.-D., et al. Evidence for circulating bone marrow-derived endothelial cells. Blood (1998) 92:362–367.[Abstract/Free Full Text]
  8. Lin Y., Weisdorf D.J., Solovey A., Hebbel R.P. Origins of circulating endothelial cells and endothelial outgrowth from blood. J. Clin. Invest. (2000) 105:71–77.[Web of Science][Medline]
  9. Peichev M., Naiyer A.J., Pereira D., et al. Expression of VEGFR-2 and AC133 by circulating human CD34+ cells identifies a population of functional endothelial precursors. Blood (2000) 95:952–958.[Abstract/Free Full Text]
  10. Gehling U.M., Ergün S., Schumacher U., et al. In vitro differentiation of endothelial cells from AC133-positive progenitor cells. Blood (2000) 95:3106–3112.[Abstract/Free Full Text]
  11. Gittenberger-de Groot A.C., DeRuiter M.C., Bergwerff M., Poelmann R.E. Smooth muscle cell origin and its relation to heterogeneity in development and disease. Arterioscler. Thromb.Vasc. Biol. (1999) 19:1589–1594.[Free Full Text]
  12. DeRuiter M.C., Poelmann R.E., VanMunsteren J.C., et al. Embryonic endothelial cells transdifferentiate into mesenchymal cells expressing smooth muscle actins in vivo and in vitro. Circ. Res. (1997) 80:444–451.[Abstract/Free Full Text]
  13. Wunsch A., Little C.D., Markwald R.R. Cardiac endothelial heterogeneity is demonstrated by the diverse expression of JB3 antigen, a fibrillin-like protein of the endocardial cushion tissue. Dev. Biol. (1994) 165:585–601.[CrossRef][Web of Science][Medline]
  14. Tuder R.M., Groves B., Badesch D.B., Voekel N.F. Exuberant endothelial cell growth and elements of inflammation are present in plexiform lesions of pulmonary hypertension. Am. J. Pathol. (1994) 144:275–285.[Abstract]
  15. Beranek J.T. Vascular endothelium-derived cells containing smooth muscle actin are present in restenosis. Lab. Invest. (1995) 72:771.[Web of Science][Medline]
  16. Shimizu K., Sugiyama S., Aikawa M., et al. Host bone-marrow cells are a source of donor intimal smooth-muscle-like cells in murine aortic transplant arteriopathy. Nat. Med. (2001) 7:738–741.[CrossRef][Web of Science][Medline]
  17. Han C.-I., Campbell G.R., Campbell J.H. Circulating bone marrow cells can contribute to neointimal formation. J. Vasc. Res. (2001) 38:113–119.[CrossRef][Web of Science][Medline]
  18. Sata M., Saiura A., Kunisato A., et al. Hematopoietic stem cells differentiate into vascular cells that participate in the pathogenesis of atherosclerosis. Nat. Med. (2002) 8:403–409.[CrossRef][Web of Science][Medline]
  19. Simper D., Stalboerger P.G., Panetta C.J., et al. Smooth muscle progenitor cells in human blood. Circulation (2002) 106:1199–1204.[Abstract/Free Full Text]
  20. Yamashita J., Itoh H., Hirashima M., et al. Flk1-positive cells derived from embryonic stem cells serve as vascular progenitors. Nature (2000) 408:92–96.[CrossRef][Medline]
  21. Pesce M., Orlandi A., Iachininoto M.G., et al. Myoendothelial differentiation of human umbilical cord blood-derived stem cells in ischemic limb tissues. Circ. Res. (2003) 93.
  22. Hayashi K., Saga H., Chimori Y., et al. Differentiated phenotype of smooth muscle cells depends on signaling pathways through insulin-like growth factors and phosphatidylinositol 3-kinase. J. Biol. Chem. (1998) 273:28860–28867.[Abstract/Free Full Text]
  23. Duband J.L., Gimona M., Scatena M., Sartore S., Small J.V. Calponin and SM22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development. Differentiation (1993) 55:1–11.[CrossRef][Web of Science][Medline]
  24. Davidoff A.M., Ng C.Y.C., Brown P., et al. Bone marrow-derived cells contribute to tumor neovasculature and, when modified to express en angiogenesis inhibitor, can restrict tumor growth in mice. Clin. Cancer Res. (2001) 7:2870–2879.[Abstract/Free Full Text]
  25. De Bont E.S.J.M., Guikema J.E.J., Scherpen F., et al. Mobilized human CD34+hematopoietic stem cells enhance tumor growth in a nonobese diabetic/severe combined immunodeficient mouse model of human non-Hodgkin's lymphoma. Cancer Res. (2001) 61:7654–7659.[Abstract/Free Full Text]
  26. Lyden D., Hattori K., Dias S., et al. Impaired recruitment of bone-marrow-derived endothelial and hematopoietic precursor cells blocks tumor angiogenesis and growth. Nat. Med. (2001) 7:1194–1201.[CrossRef][Web of Science][Medline]
  27. Wang H.U., Chen Z.-F., Anderson D.J. Molecular distinction and angiogenic interaction between embryonic arteries and veins revealed by ephrin-B2 and its receptor Eph-B4. Cell (1998) 93:741–753.[CrossRef][Web of Science][Medline]
  28. Zhong T.P., Childs S., Leu J.P., et al. Gridlock signaling pathway fashions the first embryonic artery. Nature (2001) 414:216–220.[CrossRef][Medline]
  29. Shin D., Garcia-Gardena G., Hayashi S.-I., et al. Expression of EphrinB2 identifies a stable genetic difference between arterial and venous vascular smooth muscle as well as endothelial cells and marks subsets of microvessels at sites of adult neovascularization. Dev. Biol. (2001) 230:139–150.[CrossRef][Web of Science][Medline]
  30. Hellström M., Kalén M., Lindahl P., Abramsson A., Betsholtz C. Role of PDGF-B and PDGFR-β in recruitment of vascular smooth muscle cells and pericytes during embryonic blood vessel formation in the mouse. Development (1999) 126:3047–3055.[Abstract]
  31. Frid M.G., Kale V.A., Stenmark K.R. Mature vascular endothelium can give rise to smooth muscle cells via endothelial–mesenchymal transdifferentiation. In vitro analysis. Circ. Res. (2002) 90:1189–1196.[Abstract/Free Full Text]
  32. Murohara T., Ikeda H., Duan J., et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization. J. Clin. Invest. (2000) 105:1257–1536.
  33. Kalka C., Masuda H., Takahashi T., et al. Transplantation of ex vivo expanded endothelial progenitor cells for therapeutic neovascularization. Proc. Natl. Acad. Sci. U. S. A. (2000) 97:3422–3427.[Abstract/Free Full Text]
  34. Kawamoto A., Gwon H.-C., Iwaguro H., et al. Therapeutic potential of ex vivo expanded endothelial progenitor cells for myocardial ischemia. Circulation (2001) 103:634–637.[Abstract/Free Full Text]
  35. Yurugi-Kobayashi T., Itoh H., Yamashita J., et al. Effective contribution of transplanted vascular progenitor cells derived from embryonic stem cells to adult neovascularization in proper differentiation stage. Blood (2002).
  36. Vajkoczy P., Blum S., Lamparter M., et al. Multistep nature of microvascular recruitment of ex vivo-expanded embryonic endothelial progenitor cells during tumor angiogenesis. J. Exp. Med. (2003) 12:1755–1765.
  37. Bergers G., Song S., Meyer-Morse N., Bergsland E., Hanahan D. Benefits of targeting both pericytes and endothelial cells in the tumor vasculature with kinase inhibitors. J. Clin. Invest. (2003) 111:1287–1295.[CrossRef][Web of Science][Medline]

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