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Stem Cells Vol. 24 No. 11 November 2006, pp. 2420 -2427
doi:10.1634/stemcells.2005-0494; www.StemCells.com
© 2006 AlphaMed Press

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TISSUE-SPECIFIC STEM CELLS

TGFß1 Induces Vasculogenesis and Inhibits Angiogenic Sprouting in an Embryonic Stem Cell Differentiation Model: Respective Contribution of ALK1 and ALK5

Christine Malleta, Daniel Vittetb, Jean-Jacques Feigea, Sabine Baillya

aInstitut National de la Santé et de la Recherche Médicale (INSERM) EMI 01-05, Département Réponse et Dynamique Cellulaires (DRDC), Commissariat à l'Energie Atomique (CEA)-Grenoble, France;
bINSERM EMI 02-19, DRDC, CEA-Grenoble, France

Key Words. Transforming growth factor-ß1 • Embryonic stem cells • Vasculogenesis • Angiogenesis • Activin receptor-like kinase-1 • Activin receptor-like kinase-5

Correspondence: Sabine Bailly, Ph.D., INSERM EMI 01-05 DRDC/ANGIO, CEA-Grenoble, 17 rue des Martyrs, 38054 Grenoble, France. Telephone: (33) 4 38 78 92 14; Fax: (33) 4 38 78 50 58; e-mail: sbailly{at}cea.fr

Received October 6, 2005; accepted for publication July 1, 2006.

   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
Transforming growth factor-ß1 (TGFß1) is a multipotent cytokine that is involved in the regulation of vasculogenesis and angiogenesis. However, the actions of TGFß1 on vascular cells in vitro and in vivo are extremely complex and still incompletely understood. The aim of the present study was to investigate the role of TGFß1 and its two type I receptors, activin receptor-like kinase-1 (ALK1) and ALK5, in an embryonic stem cell (ESC) differentiation model that recapitulates the developmental steps of vasculogenesis and sprouting angiogenesis. We show that TGFß1 increases endothelial cell differentiation in a vascular endothelial growth factor (VEGF)-independent manner and inhibits endothelial tube formation. Furthermore, we demonstrate that undifferentiated ESCs express ALK5 but do not express ALK1, with ALK1 being expressed only after day 5 of differentiation. Finally, we demonstrate that constitutively active forms of ALK1 and ALK5 both inhibit growth factor-induced endothelial sprouting from embryoid bodies. In conclusion, the use of this ESC differentiation model allowed us to propose the following model: at early stages of development, TGFß1, through the ALK5 receptor, is provasculogenic in a VEGF-independent manner. Later, in differentiated endothelial cells in which both ALK1 and ALK5 are expressed, both receptors are implicated in inhibition of sprouting angiogenesis.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
Vascularization of organs and tissues proceeds by two related but distinct processes: vasculogenesis and angiogenesis [1]. During vasculogenesis, primitive blood vessels develop from angioblast precursor cells that differentiate into endothelial cells and assemble into cord-like vascular structures that further connect into a primary network. Angiogenesis corresponds to the formation of new blood vessels from the pre-existing blood vasculature by sprouting, splitting, and remodeling of the primitive vascular network. Both vasculogenesis and angiogenesis are involved in the development of a functional vascular system in the embryo but also contribute to postnatal blood vessel formation. In the adult, a neovascular response occurs in a variety of physiological and pathological settings, including wound healing, recovery from myocardial infarction, inflammation-related diseases, solid tumor growth, and tumor metastasis. Vasculogenesis and angiogenesis are under the tight regulation of growth factors. These factors include vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), platelet-derived growth factor, and transforming growth factor-ß1 (TGFß1).

TGFß1 is a multipotent cytokine that has been shown to be involved in the regulation of vasculogenesis and angiogenesis [2, 3]. The actions of TGFß1 on the vascular cells in vitro and in vivo are extremely complex and depend on the origin of the cells and on the experimental conditions. TGFß1 exerts its effects by interacting with two transmembrane serine/threonine kinases, known as type I and type II receptors. TGFß1 binds to the type II receptor that recruits and phosphorylates a type I receptor to produce heteromeric-signaling complexes. In most cell types, TGFß1 signals through the transforming growth factor-ß type I receptor (TGFßRI), also known as activin receptor-like kinase-5 (ALK5). In endothelial cells, however, recent studies have shown that TGFß1 can bind to and transduce signals through both ALK1, another type 1 receptor, and ALK5 [4]. Signaling through ALK1 or through ALK5 will trigger different signaling cascades through activation of two different classes of Smads. Activated ALK5 phosphorylates Smad2 and 3, whereas activated ALK1 phosphorylates Smad1 and Smad5. These receptor-regulated Smads then form heteromeric complexes with Smad4 which translocate to the nucleus, where they control gene expression [5]. The importance of the TGFß1 superfamily members in vasculogenesis has been suggested by the findings that knockout mice deficient for TGFß1 superfamily signaling components (TGFß1, ALK5, ALK1, endoglin, Smad1, and Smad5) exhibit defects in vascular tissues [6]. Moreover, heterozygous mutations in human genes encoding endoglin, ALK1, or Smad4 cause the vascular disorder known as Rendu-Osler-Weber syndrome [7–9]. However, the lack of in vitro systems recapitulating vascular development has hampered the dissection of the respective roles of the TGFß1 signaling components in this process.

In this report, we have analyzed the role of TGFß1 and its two type I receptors, ALK1 and ALK5, in vascular development using an in vitro model of embryonic stem cell (ESC) differentiation. We have previously demonstrated that endothelial development within ESC-derived embryoid bodies (EBs) follows an orderly sequence of events that recapitulates murine vasculogenesis in vivo [10]. In addition, when cultured in a collagen I matrix, EBs can develop a network of branching endothelial outgrowths that are covered with pericytes, thus constituting a valuable model to study angiogenesis [11, 12].

Here, we show that TGFß1 increases endothelial cell differentiation and inhibits endothelial tube formation. Given that ALK5 is already expressed in undifferentiated ESCs, whereas ALK1 is absent until day 5 of differentiation, we suggest that TGFß1 induction of vasculogenesis is mediated by ALK5. In contrast, because constitutively active forms of ALK1 and ALK5 both inhibit angiogenic growth factor-induced endothelial sprouting from EBs embedded into type I collagen gels, we propose that both receptors are implicated in TGFß1-mediated inhibition of sprouting angiogenesis.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
ESC Differentiation into the Endothelial Lineage
Differentiation of subconfluent R1 ESCs [13] (a generous gift from Dr. A. Nagy, Toronto) was initiated in Iscove's medium containing Glutamax (Iscove's modified Dulbecco's medium; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 1% methylcellulose (FLUKA, Steinheim, Switzerland, http://www.sigmaaldrich.com), 15% fetal calf serum (Invitrogen), 450 µM monothioglycerol (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com), 10 µg/ml insulin (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com), 50 U/ml penicillin, and 50 µg/ml streptomycin (Invitrogen) as previously described [14]. Cultures were maintained without further feeding for up to 11 days or collected at the time indicated. TGFß1 (2 or 10 ng/ml; R&D Systems Inc., Minneapolis, http://www.rndsystems.com) and VEGF (10 ng/ml; AbCys s.a., Paris, http://www.abcysonline.com) were added at days 0, 2, 4, and 6. Neutralizing anti-VEGF antibody (0.1 µg/ml; AF-293-NA; R&D Systems Inc.) was preincubated for 1 hour prior to growth factor addition. For secondary culture in collagen gels, ESCs were differentiated as described above in the presence of a cocktail of growth factors containing 50 ng/ml human VEGF (AbCys s.a.), 100 ng/ml FGF-2 (a generous gift from Dr. A. Baird, Whittier Institute, La Jolla, CA), 2 U/ml mouse erythropoietin (Roche Diagnostics), and 10 ng/ml human interleukin-6 (AbCys s.a.) as previously described [14]. EBs were collected after 11 days of differentiation and cultured in the same medium as above supplemented with 1.25 mg/ml collagen I (BD Biosciences, San Diego, http://www.bdbiosciences.com). Different doses of TGFß1 (0.1, 0.5, 2, and 10 ng/ml; R&D Systems Inc.) were added at day 11 of differentiation at initiation of the secondary culture in collagen gel. Recombinant adenoviral vectors expressing the constitutively active form of ALK1 (AdALK1ca, mutation of glutamine 201 into aspartic acid) or ALK5 (AdALK5ca, mutation of glutamine 204 into aspartic acid) tagged with an influenza virus hemagglutinin epitope (generated by Dr. M. Fujii and kindly given to us by Dr. K. Miyazono, University of Tokyo, Tokyo) [15] were used to express ALK1ca and ALK5ca, respectively. A recombinant adenovirus-expressing bacterial ß-galactosidase (Adß-gal) was used as a control. EBs were infected at day 10 of differentiation for 15 hours at multiplicity of infection (MOI) of 500 or 1,000 as indicated. On the next day (day 11), EBs were treated as usual for the second step of differentiation.

Immunocytochemistry
Six-day-old EBs embedded in methylcellulose or 14-day-old EBs embedded in collagen I gel were fixed in methanol-dimethylsulfoxide (4:1) overnight at 4°C and stained with a rat monoclonal anti-mouse platelet/endothelial cell adhesion molecule (PECAM)/CD31 antibody (clone MEC-13.3, a gift from Dr. A. Vecchi, Milan, Italy) as previously described [16]. EBs were then mounted in aqueous medium before examination with a microscope. For quantitation of endothelial sprouting, images were captured with a digital camera and measurement of the total length of endothelial sprouts was achieved by morphometric analysis using the Visiolab@2000 software (Molecular Devices, Downingtown, PA, http://www.moleculardevices.com).

Dissociation of EBs
EBs embedded in methylcellulose were dissociated in cell-dissociation enzyme-free phosphate-buffered saline (PBS)-based buffer (Invitrogen) for 30 minutes at 37°C after dilution of the semisolid methylcellulose medium with PBS. EBs embedded in collagen I were collected after digestion of the collagen matrix by 30 minutes of treatment at 37°C with PBS solution containing 0.2% collagenase B (Roche Diagnostics).

Flow Cytometry Analysis
After two washes in PBS, cells from 3-, 4-, 5-, 6-, and 7-day-old dissociated EBs (1 x 106 cells) were incubated at 4°C with fluorescein isothiocyanate-conjugated anti-mouse PECAM/CD31 antibody (1:50; BD Pharmingen, San Diego, http://www.bdbiosciences.com/pharmingen) or phycoerythrin-conjugated anti-mouse Flk-1 antibody (1:25; BD Pharmingen) or both for 40 minutes in 2% bovine serum albumin-PBS. After three washing steps in PBS, quantitative fluorescence analysis was performed using a FACSCalibur flow cytometer and the software program CellQuest (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com). Histograms of cell number versus logarithmic fluorescence intensity were recorded for at least 10,000 cells per sample.

Reverse Transcription-Polymerase Chain Reaction Analysis
Total RNA from undifferentiated ESCs, dissociated EBs, or PECAM-negative and PECAM-positive ESCs from 11-day-old dissociated EBs cell-sorted by fluorescence-activated cell sorting (FACS) (FACStar; Becton, Dickinson and Company) was isolated using a total RNA isolation kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). First-strand cDNAs were generated using 2 µg RNA, Superscript reverse transcriptase (Invitrogen), and random hexamer primers (GE Healthcare, Little Chalfont, Buckinghamshire, UK, http://www.gehealthcare.com). For the polymerase chain reaction (PCR), first-strand cDNA (the equivalent of 40 ng of reverse-transcribed RNA) was amplified in a final volume of 20 µl with 1 U of Taq DNA polymerase (Qiagen) and 10 pmol of each primer (Table 1). After 5 minutes of initial denaturation at 94°C, reactions were cycled through 1 minute at 94°C, 1 minute of annealing at primer-specific temperature (Table 1), and 1 minute of primer extension at 72°C. Final extension was carried out for 5 minutes at 72°C. To ensure quantitative results, the number of PCR cycles for each set of primers was checked to be in the linear range of amplification. In addition, all cDNA samples were adjusted to yield equal amplification of HPRT (hypoxanthine phosphoribosyltransferase) as an internal standard. All PCR experiments included reverse transcriptase negative controls.


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  		Table 1. Oligonucleotide primers used for reverse transcription-polymerase chain reaction analysis

 
Statistical Analysis
The results were subjected to statistical analysis by the Student's t test; the level of significance was set as * p < .05 and ** p < .01.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 
TGFß1 Enhances ESC Differentiation into Endothelial Cells
In a first step, ESCs were cultured in semisolid methylcellulose in order to differentiate into endothelial cells in the presence or absence of TGFß1 (2 or 10 ng/ml) added every other day for 6 days. Representative control (CTL) and TGFß1-treated EBs are illustrated in Figure 1A. The percentage of EBs containing PECAM-positive cells was 65% ± 9%, 81% ± 14%, and 89% ± 7% in the presence of 0, 2, and 10 ng/ml TGFß1, respectively, demonstrating that TGFß1 increased the number of vascular EBs. Flow cytometry quantitation of PECAM-positive cells obtained from the dissociation of 7-day-old EBs showed that there was a significant dose-dependent increase in the percentage of PECAM-positive cells in the presence of TGFß1 (Fig. 1B). Although PECAM is widely used as a marker of endothelial cells, it was previously reported that undifferentiated ESCs express PECAM [10] but as in vitro differentiation proceeds PECAM expression decreases to be gradually re-expressed in endothelial cells [17]. To make sure that the increase in PECAM-positive cells does not result from the persistence of undifferentiated ESCs, we performed a kinetic analysis of PECAM expression in response to TGFß1 from day 3 to day 7. Figure 1C confirms the presence of a high number of ESC-derived cells expressing PECAM until day 3 and demonstrates that the PECAM increase induced by TGFß1 does not result from the presence of some PECAM-positive undifferentiated cells but is due to an increase in PECAM re-expression at day 7.


Figure 1
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  		Figure 1. TGFß1 increases the percentage of PECAM-positive cells. R1 ESCs were cultured in the presence or absence of TGFß1 (2 or 10 ng/ml) in semisolid methylcellulose medium. (A): Representative fields of PECAM whole-mount immunostaining to reveal endothelial cells of EBs grown in the absence or presence of TGFß1. (B): PECAM immunostaining of cells obtained from EBs dissociated at day 7 was quantified by flow cytometry. Results are expressed as the percentage of PECAM-positive cells harvested. Values are the mean ± SD from four independent experiments. * p < .05. (C): PECAM immunostaining of cells obtained from EBs dissociated at days 3, 4, 5, 6, and 7 of differentiation. Values are the mean of one representative experiment performed in duplicate. Abbreviations: CTL, control; EB, embryoid body; PECAM, platelet/endothelial cell adhesion molecule; TGFß1, transforming growth factor-ß1.

 
To confirm that the PECAM-positive population that we were studying corresponded to endothelial cells, we also analyzed the expression of Flk-1, the type 2 VEGF receptor, which is the earliest marker of developing endothelial cells [18]. Figure 2A shows that TGFß1 addition increased the percentage of Flk-1-positive cells. We also measured the effect of TGFß1 on the double PECAM/Flk-1-positive population, which marks cells committed to the endothelial lineage during ESC differentiation (Fig. 2B). Our data indicated that TGFß1 increased the number of PECAM/Flk-1-positive cells, confirming that TGFß1 increased ESC differentiation into endothelial cells.


Figure 2
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  		Figure 2. TGFß1 increases the percentage of Flk-1-positive and PECAM-positive/Flk-1-positive cells. R1 ESCs were cultured in the presence or absence of TGFß1 (10 ng/ml) in semisolid methylcellulose medium. Percentage of Flk-1-positive (A) or PECAM- and Flk-1-positive (B) ESCs obtained from embryoid bodies dissociated at days 3, 4, 5, 6, and 7 of differentiation quantified by flow cytometry. Values are the mean of one representative experiment performed in duplicate. Abbreviations: CTL, control; Flk-1, fetal liver kinase-1; PECAM, platelet/endothelial cell adhesion molecule; TGFß1, transforming growth factor-ß1.

 
TGFß1-Enhanced ESC Differentiation Is Not Mediated by VEGF
We next wanted to determine how TGFß1 effects related to VEGF mediated differentiation of endothelial cells. As shown in Figure 3, we found that VEGF at 10 ng/ml was as potent as TGFß1 in inducing endothelial cell formation (16% vs. 14%, respectively). Because VEGF expression is clearly increased after day 5 of differentiation as determined by quantitative real-time PCR (data not shown), we tested the effect of a VEGF blocking antibody on TGFß1-induced endothelial cell differentiation. As shown in Figure 3, addition of anti-VEGF (10 µg/ml) had no effect on control cells, completely abolished VEGF effect, and did not inhibit TGFß1-induced increase of PECAM-positive cells and even actually increased it (14%–20%). Taken together, these data show that TGFß1 is a potent inducer of endothelial cell differentiation and that this effect is not mediated through VEGF.


Figure 3
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  		Figure 3. TGFß1 induction of PECAM-positive cells is independent of VEGF. TGFß1 and VEGF were used at 10 ng/ml and anti-VEGF at 0.1 µg/ml and preincubated for 1 hour before addition to ESCs. PECAM immunostaining of cells obtained from embryoid bodies dissociated at day 7 was quantified by flow cytometry. Values are the mean ± SD of two independent experiments. ** p < .01. Abbreviations: CTL, control; PECAM, platelet/endothelial cell adhesion molecule; TGFß1, transforming growth factor-ß1; VEGF, vascular endothelial growth factor.

 
TGFß1 Inhibits Endothelial Tube Formation
We next tested the effect of TGFß1 on angiogenic growth factor-induced endothelial sprouting from EBs identified by PECAM staining. Addition of TGFß1 (10 ng/ml) had a dramatic negative effect on the angiogenic sprout formation, visualized by PECAM immunoreactivity, as illustrated in Figure 4A. Quantitative analysis revealed that the addition of TGFß1 reduced, in a dose-dependent manner, the length of endothelial sprouts induced by angiogenic growth factors (Fig. 4B).


Figure 4
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  		Figure 4. TGFß1 inhibits angiogenesis induced by angiogenic GF. Eleven-day-old embryoid bodies (EBs) were cultured for 3 days in type I collagen gel in the absence or presence of TGFß1 (0.1, 0.5, 2, and 10 ng/ml). (A): PECAM whole-mount immunostaining to reveal endothelial cells. (B): Quantitative analysis of the EB mean total PECAM-positive sprout length was performed on at least 35 EBs. Data represent mean values of two independent experiments ± SEM. **p < .01. Abbreviations: GF, growth factor; PECAM, platelet/endothelial cell adhesion molecule; TGFß1, transforming growth factor-ß1.

 
Kinetics of TGFß1 Receptor Expression During Vasculogenesis and Angiogenesis
To study TGFß1 receptor expression, reverse transcription-PCR analysis was performed on RNA extracted from ESC-derived EBs collected from day 0 to day 14 (Fig. 5A). The type I TGFß receptor ALK5 was present both in undifferentiated ESCs and throughout the differentiation process. In contrast, the other TGFß type 1 receptor, ALK1, was not expressed in undifferentiated ESCs. Its expression started at day 6 of differentiation (in fact, we could detect it at day 5; data not shown). The type 2 TGFß1 receptor (TßRII) was not expressed in undifferentiated ESCs but rapidly appeared after 2 days of differentiation. The two endothelial markers were also studied; FLK-1 gene expression was found to be rapidly upregulated whereas PECAM mRNA was already expressed in undifferentiated cells then disappeared and reappeared by day 6, in accordance with previous studies [10]. We also investigated the effect of TGFß1 on ALK1 and ALK5 mRNA expression and found that TGFß1 did not modify the expression of these two receptors (data not shown). Finally, we analyzed TGFß receptor expression profiles in the endothelial population after 11 days of differentiation, which is at the start of the secondary culture in collagen gels. To this end, PECAM-positive and PECAM-negative cells were sorted by FACS. PECAM-negative cells expressed ALK5 and TßRII, whereas PECAM-positive cells, which correspond to endothelial cells, expressed ALK5, TßRII, and a high-level of ALK1 (Fig. 5B).


Figure 5
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  		Figure 5. Semiquantitative RT-PCR analysis of gene expression in ESC-derived embryoid bodies (EBs). (A): RNA was extracted from undifferentiated ESCs (Day 0 [D0]) and from EBs at the day indicated and analyzed for the expression of ALK5, ALK1, TßRII, FLK-1, PECAM, and HPRT. HPRT was amplified to normalize for the amount of RNA used as starting material. RT- are negative controls performed on an RNA sample that have not been reverse-transcribed. Shown are data from one typical experiment performed in duplicate. (B): RNA was extracted from PECAM-negative and PECAM-positive ESCs that were cell-sorted by fluorescence-activated cell sorting at day 11 of differentiation. ALK1, ALK5, TßRII, and HPRT expressions were analyzed by RT-PCR. Abbreviations: ALK1, activin receptor-like kinase-1; ALK5, activin receptor-like kinase-5; D, day; FLK-1, fetal liver kinase-1; HPRT, hypoxanthine phosphoribosyltransferase; PECAM, platelet/endothelial cell adhesion molecule; RT-PCR, reverse transcription-polymerase chain reaction; TßRII, transforming growth factor-ß type II receptor.

 
Expression of Constitutively Active Forms of ALK1 or ALK5 Inhibits Endothelial Tube Formation
To study the respective roles of ALK1 and ALK5 in TGFß1-induced inhibition of angiogenesis, we studied the effect of the expression of the constitutively active forms of these two receptors (ALK1ca and ALK5ca) on the angiogenic step. To do this, 10-day-old EBs were infected with adenoviruses expressing either ß-gal as a control or ALK1ca or ALK5ca at an MOI of 500 or 1,000. EBs were then plated in collagen gels, and the length of PECAM-positive sprouts was counted 4 days later. We found that ALK1ca and ALK5ca significantly reduced, in a dose-dependent manner, the length of endothelial sprouts elicited by angiogenic growth factors in the collagen gel (Fig. 6). Coexpression of the two ca receptors (MOI 500 + 500) had a similar effect to the addition of either ALK1ca or ALK5ca at an MOI of 1,000.


Figure 6
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  		Figure 6. ALK1ca and ALK5ca expression inhibits angiogenesis. Ten-day-old embryoid bodies (EBs), cultured in methylcellulose, were infected with AdALK1ca, AdALK5ca, or Adß-gal (multiplicity of infection = 500 or 1,000) for 15 hours and were then cultured for 3 days in type 1 collagen gel in the presence of angiogenic growth factors. Endothelial sprouting was visualized by whole-mount PECAM immunostaining. The mean total length of endothelial sprouts per EB was measured by quantitative microscopy analysis performed on at least 55 EBs for each condition. Data represent mean values of two independent experiments ± SEM. ** p < .01. Abbreviations: AdALK1ca, adenovirus-expressing ALK1-ac; AdALK5ca, adovirus-expressing ALK5ca; Adß-gal, adenovirus-expressing bacterial ß-galactosidase; ALK1ca, constitutively active ALK1; ALK5ca, constitutively active ALK5; PECAM, platelet/endothelial cell adhesion molecule.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Conclusion
 Disclosures
 Acknowledgments
 References
 
The aim of this work was to define the role of TGFß1 and its receptors (ALK1 and ALK5) in vasculogenesis and angiogenesis by using a model derived from ESCs that recapitulates the early steps of vascular development: vasculogenesis and angiogenesis. With this in vitro model, we show that addition of TGFß1 has two distinct effects on vascular development. First, it increases the number of cells committed to the endothelial lineage, which indicates a provasculogenic effect. Second, TGFß1 decreases the length of the endothelial sprouts elicited by angiogenic growth factors, a step corresponding to sprouting angiogenesis. Because it is the only receptor expressed in the early stages of ESC differentiation, ALK5 is probably involved in the induction of vasculogenesis. In the angiogenic step, as both ALK1 and ALK5 are present and as expression of both ALK1 and ALK5 constitutively active forms inhibit endothelial cell sprouting, both receptors may individually or collectively be responsible for TGFß1 inhibition of sprouting angiogenesis. These findings may help reveal how TGFß1 can function as both a promoter and an inhibitor of vasculogenesis and angiogenesis, respectively.

In the present work, we evaluated the effect of TGFß1 on endothelial cell differentiation by measuring the percentage of PECAM, Flk-1, or PECAM- and Flk-1-positive cells. Although PECAM is expressed in undifferentiated ESCs, as previously shown, PECAM expression gradually decreased during ESC differentiation and completely disappeared near day 4 before being re-expressed in distal differentiation stages. This particular PECAM pattern of expression has recently been demonstrated to reflect the differential expression of PECAM isoforms [17]. We also analyzed expression of another endothelial marker, Flk-1, the type 2 VEGF receptor, which is the earliest marker of developing endothelial cells [18]. We found that, in accordance with previous work [10, 18], Flk-1 is not expressed in undifferentiated ESCs and appeared between days 3 and 4 of differentiation. Then, as demonstrated previously [18], and confirmed here (Fig. 2), a fraction of Flk-1 cells rapidly lose expression of Flk-1. However, the cells that maintained the expression of Flk-1 concomitantly started to express PECAM, demonstrating the differentiation into endothelial lineage. In the present work, we show that the addition of TGFß1 increases the PECAM-positive population and that this is not due to a persistence of PECAM-positive undifferentiated ESCs (Fig. 1). We also demonstrate that TGFß1 increases the Flk-1-positive population by stabilization of the Flk-1-positive population (Fig. 2A) and that it also increases the double PECAM/Flk-1-positive population (Fig. 2B). Taken together, these data demonstrate that TGFß1 is an inducer of endothelial cell differentiation. This result is in accordance with a previous study showing that PECAM mRNA levels were increased in wtTßRII EBs versus {Delta}TßRII EBs [19] and corroborates in vivo data obtained with TGFß1-deficient mice that showed defective vasculogenesis due to a defect in endothelial differentiation [20]. Accordingly, Gualandris et al. [21] demonstrated that latent TGFß-binding protein-1 promotes in vitro differentiation of ESCs toward the endothelial lineage. The comparison of the effects of TGFß1 with those of VEGF, a crucial factor for vasculogenesis [22, 23], demonstrates that TGFß1 is nearly as potent as VEGF. Given that VEGF is already expressed by day 6 of differentiation in ESCs [24] and that there is evidence suggesting that VEGF could be a target for TGFß1 [25], we tested whether TGFß1 mediated its effects through VEGF using a blocking anti-VEGF antibody. We demonstrated that the anti-VEGF antibody did not inhibit TGFß1 activity but rather increased its effects. Although surprising, this latter point is in accordance with the notion that VEGF is not the only differentiating factor necessary for endothelial cells. Indeed, although vessel formation is disrupted, differentiation of endothelial cells is normal in mice or ESCs that lack VEGF [22–24]. Further work will be required to understand the mechanisms by which TGFß1 induces endothelial cell differentiation.

In the present work, we also studied the effect of TGFß1 on angiogenic sprouting by measuring the length of PECAM-positive sprouts. Although PECAM expression can be observed on some nonendothelial cells at this stage of differentiation (like hematopoietic precursors), PECAM has proven to be a valuable tool to identify endothelial cells at this differentiation stage [11, 14]. Indeed, several reports mentioned that hematopoietic precursor development within EBs involved a very limited number of cells given that they accounted in the absence of hematopoietic growth factors for less than 1% of the total EB cell population at day 10 of differentiation [10, 26]. In addition, we previously established that PECAM-positive sprouts arising from 11-day-old EBs embedded into collagen 1 gels are generated by endothelial cells and are of endothelial nature given that they coexpress other endothelial antigens such as VE (vascular endothelial)-cadherin and von Willebrand factor [11]. In contrast to the provasculogenic effect of TGFß1, we demonstrate here that the addition of TGFß1 inhibits endothelial angiogenic sprouting. In agreement with our work, a recent study has demonstrated that TGFß1 and activin both inhibited proliferation and sheet formation of purified ESC-derived Flk-1-positive cells [27]. The ESC/EB model corresponds to a three-dimensional in vitro assay and therefore confirms that TGFß1 is a potent inhibitor of endothelial cell invasion of gels (reviewed in [3]). Angiogenesis consists of two phases [1]: a phase of activation associated with endothelial cell migration and proliferation, which is inhibited by TGFß1, and a phase of resolution. Once a new vessel has formed, TGFß1 may promote the phase of resolution by inducing endothelial cell quiescence (by inhibiting proliferation and migration) and vessel maturation (through the deposition and organization of a basement membrane and the recruitment of pericytes). Therefore, in the present work, given that TGFß1 decreases endothelial sprouting, it is difficult to draw conclusions about the global effect of TGFß1 on angiogenesis because it will have different functions on vessel formation at different stages of the angiogenic process.

To better understand the mechanisms of TGFß1 induction of vasculogenesis and inhibition of angiogenesis, we asked whether these effects were mediated through the different TGFß type I receptors. In the present study, we found that undifferentiated ESCs express ALK5 but do not express TßRII and ALK1. TßRII is rapidly increased by day 2 of differentiation. ALK1 is expressed only after day 5 of differentiation (i.e., at the same time as PECAM re-expression and slightly later than Flk-1, two early markers of the endothelial lineage when coexpressed). These results therefore strongly suggest that the provasculogenic effect of TGFß1 on undifferentiated ESCs can probably be attributed to ALK5. Interestingly, ALK1 expression followed Flk1 expression and PECAM re-expression, confirming that ALK1 is specifically expressed in endothelial differentiated cells. Concerning the inhibitory effect of TGFß1 on the angiogenic step, because both receptors are present by day 5 of differentiation, we studied whether the expression of either of their constitutively active forms could reproduce TGFß1-induced endothelial inhibition of sprouting. ALK1ca or ALK5ca expression inhibited, in a dose-dependent manner, the formation of these sprouts, and their coexpression did not lead to an additive effect. From these data, we cannot conclude whether these inhibitory effects are direct (infected endothelial cells cannot form sprouts) or indirect (other infected cells inhibit endothelial cell sprouting). It is established that ALK1 signals through Smad1/Smad5, whereas ALK5 signals through Smad2/Smad3 and that each receptor induces a different set of genes with some overlap. In particular, it was shown that ALK1 activates the expression of Smad6 and Smad7, STAT1, and the inhibitor of DNA binding-1 (Id1) and -2 (Id2), whereas ALK5 activates that of PAI-1, SM22{alpha}, and LTBP1 [28]. Analysis of ALK1- and ALK5-regulated genes suggested that ALK5 might be more important than ALK1 in the modulation of extracellular matrix and differentiation of periendothelial cells whereas ALK1 is more involved in vascular maturation through activation of intracellular regulators. Our hypothesis therefore would be that ALK1 and ALK5, through different targets, are both involved in the formation of endothelial sprouts. This would explain the lack of redundancy between the phenotypes of ALK1- and ALK5-deficient mice, although these two types of knockout mice exhibit vascular defects with certain similarities.


   CONCLUSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
Disclosures
 Acknowledgments
 References
 
Taken together, the use of the ESC differentiation model permitted us to dissect the function of TGFß1 and the respective roles of its two receptors in vasculogenesis and sprouting angiogenesis. Our present data allow us to propose the following model: At early stages of development, TGFß1, through its ALK5 receptor, is provasculogenic in a VEGF-independent manner. In differentiated endothelial cells, where both ALK1 and ALK5 receptors are expressed, both receptors are implicated in inhibition of sprouting angiogenesis through both common and specific targets, ALK5 being more implicated in the regulation of extracellular matrix formation [29], whereas guidance of endothelial cells would be regulated by ALK1, as previously proposed [30, 31].


   DISCLOSURES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
Acknowledgments
 References
 
The authors indicate no potential conflicts of interest.


   ACKNOWLEDGMENTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
References
 
We thank Dr. M. Fujii (University of Tokyo, Japan) for providing ALK1ca and ALK5ca adenoviruses, the Vector Core of the University of Nantes supported by the Association Française contre les Myopathies (AFM) for amplification of the adenovirus vectors, and Dr. J. LaMarre (University of Guelph, Ontario, Canada) for his review of the manuscript and helpful discussions. The work was supported by INSERM (EMI 01-05), CEA (DSV, DRDC/ANGIO), and an EU grant (number QLRT-2000-01302) for the T-Angiovasc project.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Conclusion
 Disclosures
 Acknowledgments
 References
 

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