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Cardiovascular Research 2003 59(2):479-487; doi:10.1016/S0008-6363(03)00433-4
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Copyright © 2003, European Society of Cardiology

Involvement of cyclin-dependent pathway in the inhibitory effect of delphinidin on angiogenesis

Laure Favot, Sophie Martin, Thérèse Keravis, Ramaroson Andriantsitohaina and Claire Lugnier*

Pharmacologie et Physico-Chimie des Interactions Cellulaires et Moléculaires, UMR CNRS 7034, Université Louis Pasteur de Strasbourg, 74 Route du Rhin, B.P. 24, 67401 Illkirch, France

* Corresponding author. Tel.: +33-390-244-264; fax: +33-390-244-313. claire{at}aspirine.u-strasbg.fr

Received 14 October 2002; accepted 23 April 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Epidemiologic studies have shown that a diet rich in fruits and vegetables has a beneficial preventive effect for cancer and cardiovascular diseases. The mechanisms of these beneficial effects are not known although there is evidence that polyphenolic compounds in food may be of some benefit. The purpose of this study was to define the effect of delphinidin, a vasoactive polyphenol belonging to the class of anthocyanin, on endothelial cell proliferation and migration as well as on in vivo angiogenesis. Methods and results: Vascular endothelial growth factor-stimulated human umbilical endothelial cell migration and proliferation are potently inhibited by delphinidin. Flow cytometric analysis demonstrates that delphinidin inhibition of proliferation is correlated with the blockade of cell cycle in G0/G1 phase. Western blot analysis shows that delphinidin reverses the vascular endothelial growth factor-induced decrease in expression of cyclin-dependent kinase inhibitor p27kip1 and the vascular endothelial growth factor-induced increase of cyclin D1 and cyclin A, both being necessary to achieve the G1-to-S transition. Furthermore, delphinidin inhibits neovascularisation in vivo in chorioallantoic membrane model. Conclusion: Delphinidin overcomes in vitro and in vivo angiogenesis and thus appears promising for the development of an anti-angiogenic therapy.

KEYWORDS VEGF, vascular endothelial growth factor; bFGF, fibroblast growth factor; HUVEC, human umbilical vein endothelial cell; RWPC, red wine polyphenolic extract; cdk, cyclin dependent kinase; cki, cyclin kinase inhibitor of cyclin dependent kinase; DMSO, dimethylsulfoxide; HS, human serum; CAM, chorioallantoic membrane; L-NA, NG-L-nitroarginine; Delph, delphinidin


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
There is increasing evidence from epidemiologic studies that a diet rich in fruits and vegetables has a beneficial preventive effect for a variety of cancers and cardiovascular diseases. The mechanisms of these beneficial effects are not known although there is evidence that polyphenolic compounds found in grapes, wine, green tea and various fruits may provide some benefits [1–3]. This last decade, polyphenols have received significant attention for their anti-tumor and anti-atherosclerotic effects [4–9]. In many instances, these effects can be attributed to biochemical mechanisms including enhanced apoptosis, growth arrest at one or more checkpoints of cell cycle, inhibition of DNA synthesis and modulation of signal transduction pathways related to altered expression of key enzymes [5].

Angiogenesis is defined as the formation of new blood vessels from pre-existing ones and is active in physiological conditions such as embryonic development, menstrual cycle and wound healing. Angiogenesis is important for the progressive growth of solid tumors and also permits the shedding of metastasis from the primary site. In tumor-associated angiogenesis, angiogenic factors secreted by tumors such as basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), stimulate endothelial cells to degrade the vascular basal membrane and to proliferate and migrate into surrounding tissues towards the tumor mass promoting the proliferation of solid tumors [10]. Serious diseases such as tumors and metastasis, hemangiomas, atherosclerosis, diabetes are characterized by the pathological growth of new capillaries. Consequently, treatment with angiogenic inhibitors seems to be a powerful strategy to prevent the development of these pathologies [11,12]. Among polyphenols, anthocyanins have been associated with potentially beneficial effects on various diseases, such as diabetic retinopathy and various microcircular deficiencies, as well as having anti-inflammatory and chemoprotective properties [13]. Previous studies from our laboratory have shown that red wine polyphenolic compound (RWPC) treatment decreases aortic stiffness in an hypertensive model [14] and that among various anthocyanins present in RWPC, delphinidin (Fig. 1) presents the most potent vasodilatory effects [15]. In view of the main role of angiogenesis in cancer and cardiovascular diseases [16], we have studied the effect of delphinidin on angiogenesis.


Figure 1
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  		Fig. 1 Structure of delphinidin.

 
In the present study, we demonstrate that delphinidin inhibits in vivo angiogenesis as well as endothelial cell migration and proliferation. Furthermore, we show that delphinidin inhibits cell cycle progression via the modulation of the cyclin dependent kinase (cdk) cyclin kinase inhibitor of cdk (cki) machinery; the latter also being implicated in endothelial cell migration. All these data suggest that delphinidin, effective in two major steps of angiogenesis in vitro as well as in neovascularisation in vivo, may represent an anti-angiogenic compound.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
The investigation conforms 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).

2.1 Drugs and chemicals
Cell culture products were from BioWhittaker. Human serum (HS) was from PromoCell. Recombinant human VEGF was from Cell Concepts. Enhanced chemiluminescence (ECL) assay kit, Hybond-P poly(vinylidene fluoride) (PVDF) membrane and Kodak Biomax light chemiluminescence films were from Amersham Biosciences. The primary antibodies were obtained from Interchim (Montluçon, France). Horseradish peroxidase (HRP)-conjugated secondary antibodies were from Promega. Fertilized White Leghorn chicken eggs were from ‘La Ferme Avicole François Hass’ (Kaltenhouse, France). Delphinidin (chloride form) was purchased from Extrasynthèse (Genay, France). Delphinidin was diluted in 10% dimethylsulfoxide (DMSO). The final concentration of DMSO in experiments never exceeded 0.1%. Control groups received the vehicle alone. All other products were from Sigma.

2.2 Cell isolation and culture
Freshly delivered umbilical cords were obtained from a nearby hospital. Human umbilical vein endothelial cells (HUVECs) were obtained as previously described [17] and grown on plastic flasks coated with 60 mg/l type I collagen and 5 mg/l human fibronectin in M199/RPMI 1640 (50:50, v/v) containing 2 mM/l ultraglutamine I, 105 U/l penicillin, 100 mg/l streptomycin, 2.5 mg/l fungizone supplemented with 15% HS. Cells were used at the second passage.

2.3 Migration assay
Migration assay was performed as previously described by Scherberich et al. [18]. HUVECs were seeded in 35 mm diameter Petri-dishes and grown to confluence in cell culture medium supplemented with 15% HS. After 24 h of serum starvation (1% HS), a rectangular lesion was made using a cell scraper. Cells were rinsed three times with culture medium without serum and incubated with the respective experimental medium. After 24 h of migration, three selected non-adjacent fields at the lesion border were acquired using a 10x phase objective on an inverted microscope (Olympus IMT2; Tokyo, Japan) equipped with a CCD camera (Panasonic). In each field, the distance between the margin of the lesion and the most distant point of migrating cells was analyzed for the 10 most mobile cells. Analysis was made using the ‘UTHSCSA Image Tool’ program available from the internet by anonymous FTP from ftp://maxrad6.uthscsa.edu.

2.4 Cell proliferation assay
HUVECs (12,000 cells/well) were seeded on 24-multiwell plates in cell culture medium supplemented with 15% HS and allowed to attached for 4–5 h. Fifteen percent HS medium was then replaced by 1% HS medium. After a 24 h incubation, medium was removed and replaced with 500 µl/well of 1% HS medium containing test substances. Cells were allowed to proliferate for 3 days without or with 10 ng/ml of VEGF. Cell number was determined by a colorimetric assay using the ‘CellTiter 96 AQueous One Solution Cell Proliferation Assay’ from Promega [19].

2.5 Cell cycle analysis
HUVECs (9,000 cells/cm2) were seeded in Petri dishes, allowed to attached for 4–5 h and cell culture medium was then replaced with cell culture medium supplemented with 1% HS. After 24 h incubation, medium was removed and replaced with 1 ml/well of culture medium supplemented with 1% HS and containing test substances. Cells were harvested after 18 h and fixed with 70% (v/v) ethanol for at least 2 h at 4°C. Cellular DNA staining was performed in phosphate-buffered saline (PBS) containing 50 µg/ml propidium iodide and 0.1 mg/ml of RNase I for at least 30 min before analysis. Cell cycle analysis was performed by flow cytometry with CellQuest software (Becton Dickinson, San Jose, CA, USA). Data of 20,000 cells were analyzed by ModFit II (Becton Dickinson).

2.6 Western blot analysis
HUVECs were treated as described for cell cycle analysis. Cells were harvested after 18 h and lysed for 2 h at 4°C in PBS containing 0.5% Triton X-100, 1 mM Pefabloc, 2 µg/ml aprotinin, 2 µg/ml leupeptin and 2 µg/ml pepstatin. Protein concentration was determined following Lowry et al. [20]. Protein samples (30 µg) were denaturated and solubilized by heating for 5 min at 95°C in Laemmli buffer, electrophoresed on sodium dodecyl sulfate (SDS)–10% polyacrylamide gel and transferred to PVDF membranes, as described previously [21]. Membranes were processed for immunoblotting with the primary antibody (anti-cyclin D1 and anti-cyclin A at 1/1,000; anti-p27kip1 at 1/500), followed by HRP conjugates as secondary antibody (1/60,000). The immobilized antigens were detected by chemiluminescence using the ECL assay kit. Blots were scanned and densitometric analysis (relatively to their respective β-actin band) was performed using Scion Image-Release Beta 4.02 software (http://scioncorp.com).

2.7 The chicken embryo chorioallantoic membrane (CAM) assay
Fertilized chick eggs were incubated according to Auerbach et al. [22]. After 5 days of incubation, delphinidin was applied as a solution containing 2 µg to 50 µg in a final volume of 20 µl of PBS, on an area of 1 cm2 of the CAM, restricted by a plastic ring. Control and treated conditions were performed on the same egg. After 2 days of incubation, CAMs were observed in ovo under Nikon microscope (SMZ1000), and were photographed.

2.8 Statistical analysis
Results are expressed as mean±S.E.M. of n separate experiments. Analysis of variance (ANOVA) was used for statistical analysis, with P<0.05 being considered significant. Correlation coefficients were calculated by using GraphPad Prism software, San Diego, CA, USA.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Effect of delphinidin on VEGF-stimulated HUVEC migration
Migration of endothelial cells, which allows cells to disseminate from the pre-existing vessel to form new vessels, contributes to angiogenesis. We studied the effect of delphinidin on HUVEC migration only after VEGF stimulation, since in the control condition, HUVEC migration is very low (data not shown). In view of previous results from our laboratory [15,23] showing clearly that delphinidin is effective at 10 µg/ml (26 µM), we studied the anti-migratory effect of delphinidin in this range of concentration on VEGF-induced HUVEC migration. As shown in Fig. 2, delphinidin concentration-dependently inhibited HUVEC migration, by 17±2.2%, 30±2.9% and 50±2.4% for 5, 10 and 20 µg/ml, respectively (correlation coefficient r = 0.9917, P = 0.0042).


Figure 2
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  		Fig. 2 Effect of delphinidin on VEGF-stimulated cell migration. Cell monolayer was wounded and treated with 10 ng/ml VEGF and without or with 5, 10, 20 µg/ml (A) delphinidin (Delph) (the black line indicates the border of the lesion). Cell migration was photographed at 24 h and analyzed (B) as described in Methods. Data represent four experiments. ## P<0.01 versus VEGF-treated cells ### P<0.001 versus VEGF-treated cells.

 
3.2 Effect of delphinidin on basal and VEGF-stimulated HUVEC proliferation
Since proliferation of endothelial cells represents a critical step in angiogenesis, the effect of delphinidin on proliferation was investigated. Fig. 3 illustrates the antiproliferative effect of delphinidin in control and VEGF-treated cells. As expected, VEGF increased basal-HUVEC proliferation by 46±7%. Delphinidin was able to decrease in a concentration-dependent manner basal- (r = 0.999, P<0.0001) as well as VEGF-stimulated proliferation of HUVECs (r = 0.9952, P = 0.0048). In both cases, 10 µg/ml was the minimal concentration able to significantly decrease both basal- and VEGF-stimulated proliferation by 22±3.9% and 21±5.4%, respectively. At 10 µg/ml, delphinidin mainly abolished the proliferative effect of VEGF. At 20 µg/ml, delphinidin inhibited cell proliferation by 44±1.5% in control and 49±2.7% in VEGF-treated cells. We therefore choose for biochemical studies the concentration of 10 µg/ml delphinidin, this concentration being effective in VEGF-stimulated cells.


Figure 3
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  		Fig. 3 Effect of delphinidin on basal and VEGF-stimulated proliferation. HUVECs were incubated for 72 h with or without 5, 10 and 20 µg/ml delphinidin (Delph) without or with 10 ng/ml VEGF. Data represent four experiments. ** P<0.01 versus control cells, ## P<0.01 versus VEGF-treated cells.

 
Since it is well known that VEGF signaling in endothelial cells is mediated by Akt-eNOS, we investigated the effect of NG-L-nitroarginine (L-NA, 200 µM) on the inhibitory effect of delphinidin on HUVEC proliferation. The results show that L-NA treatment did not change significantly the basal and VEGF-stimulated proliferation, nor the inhibitory effect of delphinidin on basal and VEGF-stimulated proliferation (not illustrated, n = 3). These data indicate that firstly VEGF-induced proliferation of HUVECs does not involve NO pathway under the experimental conditions used, and secondly that NO signaling is not implicated in the delphinidin effect both on control and VEGF-induced HUVEC proliferation.

3.3 Effect of delphinidin on HUVEC progression in cell cycle
To investigate whether delphinidin-induced cell growth inhibition could be related to alterations in cell cycle, we evaluated the effect of delphinidin at 10 µg/ml on cell cycle distribution (Fig. 4). In agreement with proliferation studies, VEGF treatment significantly increased by 52±2.2% cell numbers in the S phase and consequently decreased by 24±2.4% cell numbers in the G0/G1 phase. Delphinidin increased cell numbers in the G0/G1 phase by 17±0.4% in the basal condition and by 25±1.1% in the VEGF-stimulated condition. These effects were associated with a 37±1.6% decrease in cell numbers in the S phase under both conditions. In the G0/G1 and S phases, delphinidin treatment nearly completely abolished the effect of VEGF. Delphinidin did not alter the percentage of cells in the G2/M phase in control as well as in VEGF-stimulated conditions. Furthermore, under any experimental conditions used in the present study, delphindin was not able to induce apoptosis in HUVECs.


Figure 4
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  		Fig. 4 Effect of delphinidin on cell cycle progression. Following growth arrest, HUVECs were treated with or without 10 µg/ml delphinidin (Delph) without or with of 10 ng/ml VEGF. The effect of treatments are shown on: cell distribution into the cell cycle phases G0/G1 (shade), S (dots) and G2/M (lines) (A); cell number in cell-cycle phases (B). Data represent four experiments. ** P<0.01 versus control cells, ## P<0.01 versus VEGF-treated cells.

 
3.4 Effect of delphinidin on p27kip1, cyclin A and cyclin D1 expression
Since delphinidin treatment of HUVECs resulted in cell cycle arrest in the G1 phase, we examined the effect of delphinidin on cell cycle regulatory molecules which are operative in the G1 phase and G1/S transition (Fig. 5).


Figure 5
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  		Fig. 5 Effect of delphinidin on protein expression of p27kip1 (A), cyclin A (B) and cyclin D1 (C). As detailed in Methods, cells were treated without or with 10 ng/ml VEGF and 10 µg/ml delphinidin (Delph). The blots shown are representative of four experiments. Histograms showing densitometric analysis are representative of four separate experiments. * P<0.05 versus control cells; ** P<0.01 versus control cells, # P<0.05 versus VEGF-treated cells, ## P<0.01 versus VEGF-treated cells.

 
We assessed the effect of delphinidin on the induction of p27kip1 which is known to inhibit the activity of cdk2/cyclin A and cdk4/cyclin D1 complexes. Western blot analysis (Fig. 5) revealed that: (i) VEGF induced a 46±10% decrease of p27kip1 expression; (ii) delphinidin had no significant effect on p27kip1 expression in basal condition but restored p27kip1 expression level in VEGF stimulated condition (Fig. 5A). We also characterized the effect of delphinidin on cyclins A and D1, both cyclins allowing cell cycle progression from S to G2/M and from G0/G1 to S, respectively. Delphinidin decreased cyclin A expression in control and VEGF-stimulated conditions, by 37±3.6% and 62±13%, respectively (Fig. 5B). The increase of cyclin A level induced by VEGF was reduced by delphindin towards control. In contrast, delphinidin had no significant effect on cyclin D1 expression in control condition (Fig. 5C), but decreased by 47±16% cyclin D1 expression in VEGF-stimulated condition, thus maintaining cyclin D1 level in its basal state.

3.5 Delphinidin effect on angiogenesis of developing embryo
To study the in vivo effect of delphinidin, we examined its effect on angiogenesis using the CAM assay (Fig. 6). Below 10 µg per embryo (b), delphinidin had no significant effect on vascular network. In doses ranges of 10–50 µg/embryo, delphinidin was able to decrease capillary development in developing CAMs (d, f, h) and the inhibition was increasing with the dose of delphinidin. Using a high dose (50 µg/embryo), delphinidin-treated CAMs presented hemorrhagic zones showing that the new vessels were not functional. No acapillary zones were detected in the control rings treated with PBS and drug vehicle (a, c, e, g).


Figure 6
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  		Fig. 6 Anti-angiogenic effect of delphinidin on CAM. Eight-day-old chick embryo CAMs were treated with various amounts of delphinidin (Delph) as described in Methods. The capillary network was examined 2 days after treatment without or with 2 µg delphinidin (1 mg/ml) (b versus a), 10 µg (5 mg/ml) (d versus c), 25 µg (12.5 mg/ml) (f versus e) and 50 µg (25 mg/ml) (h versus g). The pictures shown are representative of five experiments.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present data demonstrate that delphinidin, an anthocyanin compound, inhibits angiogenesis in vivo. This effect is associated with inhibition of two major steps of angiogenesis: endothelial cell migration and proliferation. Delphinidin induces a down-regulation of cyclin A and D1 expression and an upregulation of p27kip1 expression, these three proteins being essential in cell cycle progression. All these effects lead to the inhibition of neovascularisation of developing embryo.

According to the present work, delphinidin clearly inhibits VEGF-induced HUVEC migration. Interestingly, it has been recently reported that p27kip1 is an important regulator of cell migration [24,25] and is increased in wounded HUVEC monolayer whereas cyclin A is downregulated [26]. Thus, the herein reported restoration of p27kip1 and cyclin A expression, overcoming the decreasing effect of VEGF, indicates a possible role of p27kip1 and cyclin A expression in the inhibitory effect of delphinidin on migration.

Proliferation studies reported here clearly show that delphinidin reduces cell number at the same level in control- and VEGF-stimulated proliferation, suggesting that delphinidin acts potently on proliferative mechanism. These data demonstrate that delphinidin treatment of HUVECs results in significant cell growth inhibition accompanied by cell cycle arrest in the G1 phase. This is in agreement with studies performed with resveratrol and quercetin, two polyphenolic compounds also known to inhibit proliferation of many cell types via a G1 phase arrest [27,28].

Thus, the involvement of the cki–cdk machinery during the induction of cell cycle arrest by delphinidin in HUVECs was investigated. It is now well established in eukaryotes, that progression through the cell cycle is governed by a family of protein kinase complexes [29,30]. Each complex is composed at least by a catalytic subunit, the cyclin-dependent kinase and its essential activating partner, the cyclin. Under normal conditions, these complexes are activated at specific intervals through series of events which result in progression of cells through the different phases of the cell cycle. During cell cycle progression, the cdk–cyclin complexes are inhibited via the binding to cki such as the cip/kip family of proteins [31]. Thus, our study was focused on the effect of delphinidin on cell cycle regulatory molecules operative in the G1 phase and during G1/S transition. The data demonstrate a significant up-regulation by delphinidin of the cki p27kip1 during G1 phase arrest of HUVECs, which abolishes the inhibitory effect of VEGF. Many studies have shown that p27kip1 may cause a blockade of G1/S transition, resulting in G1 phase arrest [32,33]. Because p27kip1 is regarded as an inhibitor of cdk2/cyclin A and cdk4/cyclin D1 complexes, the effect of delphinidin treatment was assessed on: (i) the expression of cyclin D1 which is operative early in the G1 phase following the activation of Ras-Raf-MAPK pathway and is needed to complete the G1 phase [34,35]; (ii) the expression of cyclin A which is required for the S phase transition and the control of DNA replication [36,37]. Delphinidin treatment of HUVECs was found to result in significant down-modulation of the two cyclins. Interestingly, delphinidin effect differs with the cyclin type: for cyclin D1 which is mainly implicated in G0/G1, delphinidin abrogates only the increasing effect of VEGF on cyclin D1 expression; for cyclin A which regulates mainly the S phase, delphinidin inhibits both basal- and VEGF-stimulated proliferation. Although delphinidin effects on VEGF-induced and basal proliferation appear rather similar, they are different on cell cycle protein expressions in control and VEGF-treated cells, suggesting an anti-angiogenic effect of delphinidin.

Since the VEGF signaling is known to be mediated by Akt-eNOS in endothelial cells, the involvement of NO pathway in the antiproliferative effect of delphinidin was investigated using the eNOS inhibitor, L-NA. The data are not in favor of the participation of NO on VEGF-induced proliferation of HUVECs under the experimental conditions used. Furthermore, the implication of NO signaling in the delphinidin effect both on control and VEGF-induced HUVEC proliferation appears unlikely. Nevertheless, it cannot be excluded that delphinidin may exert its effect by attenuating VEGF signal transduction, through its anti-oxidant property as recently described [38]. Given the fact that reactive oxygen species are involved in the VEGF signal transduction including VEGF mediated Akt activation, delphinidin might inhibit VEGF-induced proliferation by altering such pathway through its radical scavenger properties [39]. Further studies are needed to sort out this possibility.

The potent inhibitory effect of delphinidin on neovascularisation in the CAM model, in which VEGF receptors are expressed and VEGF is endogenously produced [40,41], extends the inhibitory effects of delphinidin, shown in vitro, to an in vivo model. The presence of hemorrhagic zones should be related to the anti-angiogenic properties of delphinidin. This anti-angiogenic effect could be seen at 10–50 µg/cm2 delphinidin, 25 µg/cm2 inducing a 100% acapillary zone. Resveratrol, another polyphenol, was shown to induce 100% avascular zones on the CAM model at 25 µg/0.16 cm2, suggesting that in vivo delphinidin may be as potent as resveratrol in inhibiting angiogenesis [8].

Recent studies concerning bioavailability of polyphenols are in agreement with their potential therapeutic effects. Anthocyanins are found in a large variety of food and are therefore ingested in considerable amounts as a constituent of the human diet (180–215 mg daily) [42]. According to the fact that the absorption rate of anthocyanins, such as delphinidin and cyanidin, was about 1% after oral administration [43], the concentration of delphinidin used in these study (i.e., 10 µg/ml, which corresponds to the consumption of six glasses of wine [44]) should be achieved in vivo. In contrast to other defined polyphenol compounds, delphinidin is also able to promote endothelium-dependent relaxation of rat aortic rings [15], to stimulate the increase in [Ca2+] in endothelial cells [23] and also to antagonize the epidermal growth receptor in human vulva carcinoma cell line A 431 [45]. All the above reported properties of delphinidin are of importance for the treatment of cardiovascular diseases including atherosclerosis.

Although further studies are needed to better understand the different mechanisms of delphinidin-induced inhibition of angiogenesis, we can conclude that delphinidin plays an important role as an in vivo anti-angiogenic compound by altering key protein expression in cell migration and cell proliferation.

Time for primary review 23 days.


   Acknowledgements
 
This work was supported by the ‘Centre National de la Recherche Scientifique’, ‘Association Régionale pour l'Enseignement et la Recherche Scientifique et Technique’ (ARERS, Reims, France) and ‘Fondation de France’ (France). L.F. was supported by a fellowship from the ‘Ligue Régionale contre le Cancer’ (Comité du Haut-Rhin; Colmar, France). S.M. is a recipient of a fellowship from the ‘Fondation pour la Recherche Médicale’ (France). We acknowledge the ‘Service Maternité du Centre Médico Chirurgical et Obstétrical de Strasbourg’ (France) for providing us with umbilical cords and Anita Anton for her skillful help in cell culture.


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

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