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Cardiovascular Research 2004 63(4):731-738; doi:10.1016/j.cardiores.2004.05.006
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Copyright © 2004, European Society of Cardiology

FrzA/sFRP-1, a secreted antagonist of the Wnt-Frizzled pathway, controls vascular cell proliferation in vitro and in vivo

Jérome Ezana, Lionel Lerouxb, Laurent Barandona,c, Pascale Dufourcqa,d, Béatrice Jasparda, Catherine Moreaua, Cécile Allièresa, Danièle Dareta, Thierry Couffinhala,b and Cécile Duplàa*,a

aInserm U441, Avenue du Haut Lévêque, 33 600 Pessac, France, and Université Victor Segalen Bordeaux 2, rue léo Saignat, 33 000 Bordeaux, France
bDepartment of Cardiology, Hôpital Haut Lévêque, 33604 Pessac, France
cDepartment of Cardiovascular Surgery, Hôpital Haut Lévêque, 33604 Pessac, France
dLaboratoire de Biochimie, Université Victor Segalen Bordeaux 2, rue léo Saignat, 33 000, Bordeaux, France

* Corresponding author. Tel.: +33-5-57-89-19-75; fax: +33-5-56-36-89-79. Email address: cecile.duplaa{at}bordeaux.inserm.fr

Received 18 July 2003; revised 11 May 2004; accepted 13 May 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 
Objective: FrzA, a member of the group of secreted frizzled related proteins (sFRP) that is expressed in the cardiovascular system, has been shown to antagonize the Wnt/frizzled signaling pathway. We have recently demonstrated its role in vascular cell growth control in vitro. In this study, we aimed to examine the mechanisms by which FrzA exerts its antiproliferative effect on vascular cells in vitro and its potential effect in vivo. Methods and results: On synchronized, growth-arrested endothelial cells (EC) and smooth muscle cells (SMC) treated with the recombinant purified FrzA protein, flow cytometry analysis showed that the recombinant FrzA protein delayed G1 phase and entry into S-phase. Western blot experiments demonstrated that the treatment of EC or SMC with FrzA was associated with a decrease in the level of the cyclins and cyclin-dependent kinases and an increase in cytosolic phospho-β-catenin levels. The FrzA-induced cell cycle delay was resolved by 24 h. C57BL/6J mice underwent surgery to produce unilateral hindlimb ischemia and empty adenoviruses (AdE) or adenoviruses coding for FrzA (AdFrzA) were injected at the time of the surgery. In AdFrzA-treated mice in the 7 days following surgery, we showed a decrease in cell proliferation, capillary density, and blood flow recovery and a reduced expression of cyclin and cdk activity in the ischemic muscle compared to that in the AdE-treated ischemic muscle. To gain insight into the pathway activated by FrzA overexpression, we showed an increase in the level of cytosolic phospho-β-catenin, a marker of β-catenin degradation, in AdFrzA-treated ischemic muscle compared to that in control AdE-treated ischemic muscle. Conclusion: We provided the first evidence that an impairment of the Wnt-Frizzled pathway, via FrzA overexpression, controlled proliferation and neovascularization after muscle ischemia.

KEYWORDS Capillary; Endothelial function; Signal transduction; Smooth muscle; Gene transfer; Growth factor


   1. Introduction
Top
 Abstract
 1. Introduction
2. Materials and methods
 3. Results
 4. Discussion
 References
 
The Wnt gene family includes numerous genes encoding highly conserved secreted glycoproteins related to the Wnt-1 proto-oncogene and to Drosophila wingless (Wg) gene products. The Wnts play key roles in differentiation, embryonic development and cell proliferation [1]. Targeted disruption of a number of Wnt genes has demonstrated their crucial role in the central nervous system, as well as renal, placental and limb development of the mouse embryo [2]. Moreover, Wnts and Wnt signaling have also been implicated in mammalian carcinogenesis [3–6]. Canonic Wnt signaling promotes the inactivation of the serine–threonine kinase GSK3-β which in turn leads to accumulation of cytosolic β-catenin. Subsequently, β-catenin interacts with Lef/tcf transcription factors contributing to specific gene expression [7].

The Wnts mediate intercellular communication by interacting with the frizzled proteins, a large family of putative transmembrane receptors homologous to the Drosophila frizzled (fz) protein [8]. The frizzled gene encodes a seven-pass transmembrane protein characterized by an extracellular N-terminal cysteine-rich domain (CRD) which may constitute part or all of the ligand-binding domain of the Wnt proteins [9]. sFRP gene encodes secreted frizzled related proteins related, at the sequence level, to the ligand binding CRD domain of the frizzled protein [10]. The members of this class of secreted proteins could compete for Wnt binding and antagonize Wnt function by interacting with Wnts or with their corresponding receptors [11].

A member of this family, FrzA, was originally isolated from bovine aortic endothelium in an attempt to identify genes specifically involved in the induction and/or maintenance of the quiescent, differentiated vascular endothelial phenotype [12]. FrzA is related to human SARP2 (or human sFRP) [13], mouse sFRP-1 [10]. FrzA has been reported to form a biochemical complex with Wnt-8 [14] and Wnt-1 [15] in vitro, and to interfere with Wnt signaling via an inhibition of secondary axis formation induced by Xwnt-8 and hWnt-2 in Xenopus embryos [16]. Our laboratory has previously demonstrated that in the cardiovascular system, FrzA or sFRP-1 were detected at high levels during embryogenesis in the developing heart and in adult aortic endothelium and media and in a majority of vessels [12,14]. We showed that FrzA mRNA was expressed in quiescent but not in exponentially growing bovine aortic endothelial cells (ECs) and that FrzA protein was capable of regulating endothelial cell (EC) growth, in vitro [12]. In agreement with these reports, studies have associated sFRP-1 down-regulation with breast carcinoma [17,18].

In this study, we examined the mechanisms by which FrzA exerts its antiproliferative effect on vascular cells in vitro. We observed that recombinant FrzA protein delayed the G1 phase and entry into S-phase of endothelial and smooth muscle cells (SMC). Treatment of EC or SMC with FrzA was associated with a decrease in the level of the cyclin D1 and E and of cyclin-dependent kinase associated cdk2 and cdk4. This effect was transient and resolved by 24 h. Using an adenovirus coding for FrzA injected at the time of hindlimb ischemia, we showed a decrease in cell proliferation and capillary density and a reduced expression of cyclin E and cdk2 activity in ischemic muscle. We showed that this effect was mediated through a β-catenin-dependent pathway.


   2. Materials and methods
Top
 Abstract
 1. Introduction
 2. Materials and methods
3. Results
 4. Discussion
 References
 
2.1. Recombinant FrzA protein and recombinant adenoviral constructs
Recombinant FrzA protein was obtained from conditioned medium (CM) of FrzA stably transfected COS-7 cells that was collected and concentrated as described [12]. As a control, we tested the effect of either fractions from CM from native COS-7 cells or fractions obtained before and after the FrzA peak elution fractions on cell cycle.

Adenoviral vectors (Ad) used in this study were serotype 5, with deletions in the E1A, E1B, and E3 regions of the Ad genome that rendered the vectors replication-deficient. AdFrzA contained a cytomegalovirus (CMV) promoter driving the FrzA cDNA [19]. The empty adenovirus AdE contained a CMV promoter but no cDNA. These adenoviruses were provided by the vector core of the University Hospital of Nantes (France) [20].

2.2. Cell culture–cell cycle analysis by flow cytometry
Human umbilical vein endothelial cells (HUVECs) and rat smooth muscle cells (SMC) were cultured in M199 medium supplemented with 10% and 20% fetal calf serum (FCS), respectively, antibiotics and 2 mM L-glutamine (Life technologies). SMC were serum starved for 24 h in M199 and HUVEC for 16 h with 1% FCS and 0.5% BSA. Cells were then washed twice and incubated at different time points with purified FrzA protein at estimated doses of 10 nM/l with fresh medium containing 10% FCS medium. BrdU incorporation and flow cytometric analyses were performed as previously described [21]. Labeled nuclei were analyzed for BrdU incorporation and propidium iodide (PI) staining using a fluorescence-activated cell sorter (FACS) analyser (ODAM-ATC 3000). Cell nuclei were gated into the respective cell cycle phases (G0/G1, S and G2/M phases) by analyzing BrdU vs. PI plots.

2.3. Western blotting
Polyclonal antibodies against cdk2 (sc-163), cdk4 (sc-260G), cyclin E (sc-481), cyclin D1 (sc-246), p21 (sc-397) and monoclonal antibody against p27 (sc-1641) were all obtained from Santa Cruz Biotechnology, monoclonal antibody against cyclin D1 from Novocastra Laboratories, P-ERK1/2 were from Amersham, polyclonal anti β-catenin, phospho-β-catenin and monoclonal anti {alpha}-tubulin from Sigma. Bound primary antibodies were detected with secondary horseradish-peroxidase-conjugated anti-mouse or anti-rabbit secondary antibodies and visualized with enhanced chemiluminescence (ECL) Western blotting system (Amersham).

The cdk2 kinase assay was performed as described [22]. The precipitated protein complexes were incubated with histone H1 (125 µg/ml), ATP 1 mmol/ml and ({gamma}-32P) ATP (5 µCi) in kinase buffer (final volume: 40 µl) for 30 min at 30 °C. Cytoplasmic β-catenin and phospho β-catenin were isolated as described [23].

2.4. Mouse model of unilateral hindlimb ischemia
The investigation conformed 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) and the local ethics committee for animal experiments approved procedures. Ten-week-old C57BL/6J mice underwent surgery to induce unilateral hindlimb ischemia as previously described [24]. To study cell proliferation, each animal received intraperitoneal injections of bromodeoxyuridine (BrdU Amersham, UK) (30 mg/kg) 24 h prior to sacrifice.

Immediately after surgery, mice were infected with AdFrzA or AdE (3.6 x 109 PFU in 18% pluronic gel). Transfection was performed both by direct intramuscular injection into the ischemic anterior tibialis muscle using a 27-gauge needle and by direct application to the ischemic muscle at the time of surgery.

Laser Doppler perfusion imaging (Moor Instrument, Wilmington, DE) was used to record serial blood flow measurements over the course of 11 days postoperatively, as previously described [24].

2.5. Necropsy examination
Animals were sacrificed with an overdose of sodium pentobarbital and anterior tibialis muscles were fixed in methanol and embedded in paraffin. Immunohistochemistry was performed as previously described [24], using a rat monoclonal antibody (mAb) against mouse CD-31 (Pharmingen San Diego, CA) or against BrdU (Harlan SERA-LAB, England). For measurement of capillary density or proliferation, two different sections were taken from each part of the muscle. Capillaries or BrdU positive cells were counted per 20 randomly chosen high-power fields on the two sections (per animals per time point). The results were calculated as capillaries per mm2.

2.6. Statistical analysis
Results were expressed as mean±S.D. Comparisons of continuous variables between two groups were performed by a one way ANOVA and subsequently, if statistical significance was observed by a two-sided paired t-test (Statview 5-1,Abacus). A value of P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
4. Discussion
 References
 
3.1. FrzA induced an increase in G1 phase
The potential transient role of FrzA in cell cycle regulation was studied on synchronized growth arrested HUVEC or rat SMC treated with the recombinant purified FrzA protein and analyzed by flow cytometry. Twelve hours later, control cells began entry into S-phase while cells treated with FrzA did not start their cycle. This effect on cycle delay was maintained at 18 h. By 24 h, after a complete cycle the difference was not maintained (Fig. 1). Similar effects and data were obtained on SMC by FACS analysis (not shown).


Figure 1
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  		Fig. 1 Effect of FrzA on cell cycle. Cell cycle analysis of HUVEC following FrzA treatment. Populations of synchronized HUVEC, stimulated by FrzA or control medium (Cont) were gated at different times of progression through the cell cycle after block removal. The percentage of cells in G1, S and G2M phases are indicated in the table, showing that FrzA treatment delays cell entrance in S phase. The result of one of three representative experiments is shown.

 
To confirm FrzA effect on a slower G1 phase, we followed the expression levels of the cyclin D1/cdk4 and cyclin E/cdk2 complex involved in G1 phase and in the transition G1 to S, respectively. Cell lysates were prepared from control and FrzA-treated cells. In HUVEC and SMC, maximal cyclin E expression was detected at 16 h after block removal (Fig. 2A and B). We noted that FrzA treatment decreased its expression after 12 h. Cyclin D1 expression in HUVEC was maximal at 12 h. In VSMC, cyclin D1 peak expression was between 16 and 20 h. FrzA treatment decreased its expression in both cell type after 12 h after cell cycle re induction. For cdk2 expression in HUVEC, the peak of expression appeared after 12 h. In VSMC extracts, we noted a peak of expression at 16 and 20 h after cell cycle reinduction. In HUVEC and in VSMC, we showed that FrzA decreased the levels of expression of cdk2 after 12 h. In HUVEC extracts, Western blot studies showed that cdk4 expression increased at 8 h with a peak at 12 h then decreased 20 h after the block removal. In VSMC, the peak of cdk4 expression was delayed compared to that in HUVEC (16 h in VSMC vs. 12 h in HUVEC). Stimulation of HUVEC by FrzA induced a strong decrease of cdk4 expression compared to that in non-treated cells at 12 until 20 h after cell cycle reentry. In VSMC, a significant decrease of cdk4 expression induced by FrzA was found at 16 and 20 h. In contrast, immunoblot analysis showed that FrzA sustained a high level of p27 in HUVEC and in VSMC at 16 and 20 h with no effect on p21 protein expression (Fig. 2A and B).


Figure 2
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  		Fig. 2 Effect of FrzA on cell cycle protein expression in HUVEC (A) or rat SMC (B). Western blots were done on cell lysates prepared from control and FrzA treated cells. Immunoblot analysis revealed that FrzA decreased cdk2, cdk4, cyclin E and cyclin D1 protein level as early as 12 h. In contrast, p27 levels were maintained under FrzA stimulation and no effect was found on p21 and phospho ERK1/2 protein expression through the cell cycle after block removal. (C) Effect of FrzA on β-catenin and phospho-β-catenin levels in HUVEC and SMC. The modulation of the cytoplasmic phosphorylated form of β-catenin was analyzed in FrzA-treated versus untreated synchronized HUVEC and VSMC. We found an increase of the level of the phospho-β-catenin under FrzA stimulation in vascular cells. Results are representative of six separate experiments.

 
FrzA effects on cyclin and cdk levels did not appear to be linked to the signal transduction pathway of the extracellular signal-regulated kinase (ERK) subfamily of MAP kinases. This pathway provides a common route leading to transcriptional regulation of genes that are crucial for cell growth. Previous studies have indicated that the induction of cyclin D1 results from the sustained activation of ERKs [25,26]. Throughout the 20-h time course of FrzA activation, ERK1/2 remained phosphorylated (Fig. 2A).

Next, we analyzed the modulation of the cytoplasmic phosphorylated form of β-catenin in FrzA-treated vs. untreated synchronised HUVEC and VSMC. We found an increase of the level of the phospho-β-catenin, marker of β-catenin degradation, under FrzA stimulation in vascular cells in vitro (Fig. 2C).

3.2. Adenovirus FrzA delivery decreased EC proliferation
To confirm the precocious FrzA control on vascular cell proliferation, we developed a strategy with adenovirus vectors injected into ischemic muscle to achieve FrzA local delivery. Mice infected with AdE or not infected (w/o Ad) served as controls. We had verified that AdFrzA led to expression of a functional FrzA protein of around 40 kDa with an effect on vascular cell proliferative activity in vitro and in vivo [19]. Injection of AdFrzA into muscle led to a peak expression of FrzA around 4 days, the expression was undetectable after 14 days (data not shown).

AdFrzA injection resulted in a significant decrease in the number of proliferating cells in comparison with AdE (Fig. 3A). The number of proliferating cells per mm2 at 3 days after the surgery was 125±28.3 for AdFrzA-treated animals vs. 486.7±30 for AdE-treated mice, P<0.001, and at 7 days 311.7±50 for AdFrzA vs. 888.3±71.7 for AdE-treated mice, P<0.001. Likewise, capillary density was significantly lower at day 3 and 7 after the surgery in AdFrzA-treated mice vs. AdE-treated mice (218.3±33.3 for AdFrzA vs. 608.3±90 for AdE at day 7, P<0.001) (Fig. 3B).


Figure 3
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  		Fig. 3 Effect of FrzA on cellular proliferation, capillary density and blood flow at 3 and 7 days after ischemia. Immediately after surgery, the ischemic anterior tibialis was infected with AdFrzA, AdE or not infected (w/o Ad). (A) Time-course evaluation of proliferative activity using BrdU immunostaining demonstrating a significant decrease in proliferating cell number in AdFrzA-treated muscle compared with AdE-treated or not treated (w/o Ad) muscle (n=5). (B) Time-course evaluation of capillary density in the ischemic muscle using CD31 immunostaining revealing a significant decrease in the capillary number at day 3 (D3) and day 7 (D7) after ischemia in AdFrzA-treated muscle versus AdE-treated or w/o Ad muscle (n=5). (C) Blood flow (expressed as the ratio of blood flow in ischemic versus normal leg) is extremely low in both groups immediately after the surgery (D0), but progressively increases over time. There was a significant delay in perfusion recovery in AdFrzA-treated mice compared to AdE treated mice at D7 P<0.001 and D11, P<0.01; n=12 in each group.

 
3.3. Overexpression of FrzA delayed blood flow recovery in the ischemic limb
Doppler blood flow ratio confirmed the delay in blood flow recovery in the ischemic hindlimbs of AdFrzA mice with significant differences with AdE-treated mice at day 7 (P<0.001) and day 11 (P<0.01) (Fig. 3C).

3.4. Overexpression of FrzA modulated cyclin expression in hindlimb ischemia
Analysis of cyclin E and D1 expression in ischemic tissue extracts from AdFrzA-, AdE-treated mice and not treated (w/o Ad) mice at days 3 and 7 directly assessed the precocious effects of FrzA on proliferation. At day 3, cyclin E and D1 were detected in AdE-treated and not treated (w/o Ad) muscle lysates although no cyclin E or a faint cyclin D1 expression were found in lysates from AdFrzA-treated muscle (Fig. 4A). Cdk2 protein level appeared lightly diminished in lysates at days 3 from AdFrzA-treated muscle compared to lysates from AdE-treated muscle; this modulation was correlated with a decrease in cdk2 activity (Fig. 4B).


Figure 4
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  		Fig. 4 Effect of FrzA on cell cycle protein expression in vivo. (A) Protein lysates from muscle infected either with AdFrzA or AdE or not infected (w/o Ad) were examined for their expression of cyclin E, cyclin D1 and cdk2 at day 3 (D3) and day 7 (D7) after ischemia. Results showed a decrease in cyclin E and D and a slight decrease of cdk2 expression induced by FrzA. (B) Cdk2 activity in ischemic anterior tibialis muscle. Cdk2 activity was decreased in AdFrzA-treated muscle as compared to AdE-treated muscle 3 days after the surgery.

 
3.5. Overexpression of FrzA modulated β-catenin pathway
To gain insight into the pathway activated by FrzA overexpression, we followed the expression of cytosolic β-catenin and phospho-β-catenin content (as markers of the canonical wnt-frizzled pathway). Protein analyses were done in hindlimb extracts of AdFrzA- and AdE-treated mice (n>3 for each group). As shown Fig. 5, cytosolic β-catenin was increased 7 days after ischemia in AdFrzA- and AdE-treated mice compared to non-ischemic mice relating the activation of the canonical wnt-frizzled pathway after ischemia. There was no difference in cytosolic β-catenin content in the AdFrzA-treated and in the AdE-treated hindlimb extracts. However, phospho-β-catenin, was consistently increased in AdFrzA-treated animals at day 7 compared to AdE-treated animal (Fig. 5). In consequence the ratio cytosolic phospho-β-catenin/β-catenin was increased under FrzA overexpression.


Figure 5
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  		Fig. 5 Modulation of β-catenin and phospho-β-catenin levels by FrzA in vivo. Cytosolic β-catenin expression was analysed before ischemia (D0) and 7 days after ischemia (D7) in muscle infected either with AdFrzA or AdE. Phospho-β-catenin, marker of β-catenin degradation, was consistently increased in AdFrzA-treated animals at D7 (experiment representative of six separate mice).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
References
 
The expression of sFRP-1 (FrzA murine orthologue) in the cardiovascular system during development and during adulthood [14] and the capacity of FrzA to regulate vascular cell growth [19] directed us to investigate the mechanisms by which FrzA exerts its antiproliferative effect on vascular cells in vitro and in vivo. This study provides the first evidence for a link between the Wnt/frizzled pathway and capillary proliferation.

We observed that recombinant FrzA protein delayed the G1 phase and entry into S-phase. The time needed to traverse G2/M remained constant in these cells. The transition from G1 to S phase is a key regulatory checkpoint in the cell cycle which is controlled by G1 cyclin and cdks [27]. Indeed, this G1-to-S delay was associated with a decrease in the level of the cyclin D1 and E and of cyclin-dependent kinase associated cdk2 and cdk4 and with a sustained level of p27. But this effect was transient and resolved by 24 h.

As control of proliferation appeared to be important in regulation of neovascularization and in vessel stabilization after ischemia, we tested the impact of FrzA in a murine hindlimb model in the early response to ischemia. In this model, the ligation of the femoral artery and the transection of the collateral vessels led to markedly reduced blood flow in the operated leg as assessed by laser Doppler imaging immediately after the surgery and a rapid endothelial cell proliferation [24]. This model has been used primarily to evaluate the effect of growth factors on neovascularization. Adenovirus mediating overexpression of FrzA in mice led to a reduction of vascular cell proliferation in the hindlimb compared to control mice that was supported by the decreased expression of cyclin E, cyclin D1 and cdk2 activity at day 3. The significant reduction of capillary density within 7 days after surgery was paralleled by a reduction of blood flow recovery in AdFrzA-treated mice.

Next, we examined the mechanisms by which FrzA may play a role in cell cycle control. Because of the proposed central role of MAPK in proliferative pathways, we examined their activation under FrzA stimulation in vitro. Impeding their function prevents cell proliferation in response to mitogenic stimulation [28]. The FrzA response did not correlate with a modification of the levels of phosphorylated ERK1/2 thus suggesting a distinct biochemical route regulating gene.

We further focused on the role of FrzA as a secreted antagonist of Wnt signaling. Studies have shown that activation of the Wnt pathway stimulates cell growth and have demonstrated that components of the cell cycle regulation machinery could be regulated through the β-catenin/Lef pathway. Interaction of Wnt family members with their respective frizzled receptor causes phosphorylation and inactivation of the serine–threonine kinase GSK-3 β and accumulation of cytosolic β-catenin [7]. Upon translocation in the nucleus, β-catenin serves as a transcriptional co activator of Tcf/lef target genes [29,30]. Cyclin D1 and the oncogene c-myc have been identified as a target in the Wnt signaling pathway [31,32]. In vitro, we and others have reported that FrzA may bind Wnt to prevent it from accessing its cell-surface receptor and to block further transduction of a defined Wnt signal as measured by cytosolic accumulation of β-catenin in vascular ECs and by Lef/Tcf activation [14,15]. We observed that ischemia induced a burst of cytosolic β catenin in hindlimb extracts of control and AdFrzA-treated mice. Interestingly, we showed that FrzA increased the phospho-β-catenin fraction and in consequence decreased the non-phosphorylated form of β-catenin. We confirmed this effect of FrzA by the studies on cultured VSMC and EC. Studies demonstrated that it is not the accumulated level of β catenin per se which is sufficient to transduce the Wnt signals but the level of the non-phosphorylated form of β-catenin [33]. Phosphorylation of β-catenin triggers its ubiquitylation and its degradation in proteasomes. Data from the study could imply that, in this model, FrzA induces a disruption of the Wnt/β catenin pathway activation, known to be a key regulator of cell proliferation. Other data strengthen the concept that a disruption of the Wnt pathway control could be essential for cell proliferation in vivo and in vitro. Down regulation of the expression of sFRP-1 was correlated with breast malignancy suggesting that sFRP-1 may act as a tumor suppressor gene [17,18]. This concept is also supported by a recent study of Blankesteijn et al. [34] in a rat model of myocardial infarction. They showed that many neovessels (EC and SMC) of the ischemic/infarcted area stained positively for β-catenin with a correlation between proliferation and β-catenin-positive EC.

Lately, we provided evidence that FrzA increased migration, differentiation and organization of EC into capillaries-like structures [19]. Using in vivo models (glioma cell xenografts and chick chorioallantoic membrane model), we demonstrated that overexpression of FrzA induced an angiogenic response. Because FrzA could delay EC proliferation and displayed angiogenic properties, its precise role in vessel growth has to be investigated in more complex models.

In conclusion, this study provided the first evidence that an impairment of the Wnt-Frizzled pathway via FrzA overexpression, controlled the early phase of neovascularisation after muscle ischemia.


   Acknowledgements
 
This work was supported by grants from the "Association pour la Recherche Contre le Cancer" and the European Vascular Genomics Network (EVGN). L.L. was a recipient for a fellowship from the "Fédération Française de Cardiologie", Paris, France.


   Notes
 
Time for primary review 14 days


   References
Top
 Abstract
 1. Introduction
 2. Materials and methods
 3. Results
 4. Discussion
 References
 

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