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[Cancer Research 66, 8501-8510, September 1, 2006]
© 2006 American Association for Cancer Research

Cell, Tumor, and Stem Cell Biology

The Role of the Vascular Endothelial Growth Factor–Delta-like 4 Ligand/Notch4-Ephrin B2 Cascade in Tumor Vessel Remodeling and Endothelial Cell Functions

Patricia Hainaud, Jean-Olivier Contrerès, Aude Villemain, Lang-Xia Liu, Jean Plouët, Gérard Tobelem and Evelyne Dupuy

Institut des Vaisseaux et du Sang, INSERM U689, IFR139, Paris VII-Denis Diderot, Hôpital Lariboisière, Paris, France

Requests for reprints: Evelyne Dupuy, Institut des Vaisseaux et du Sang, INSERM U689, IFR139, Paris VII-Denis Diderot, Hôpital Lariboisière, 8 rue Guy Patin, 75475 Paris, Cedex 10, France. Phone: 33-1-45-26-21-98; Fax: 33-1-42-82-94-73; E-mail: evelyne.dupuy{at}lrb.aphp.fr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Vascular endothelial growth factor (VEGF) and Delta-like 4 ligand (DLL4) are the only genes whose haploinsufficiency results in vascular abnormalities. Although many common pathways are up-regulated in both vascular development and tumor angiogenesis and in vascular remodeling, the role of the Delta/Notch pathway has not been clearly defined in tumor angiogenesis. In this study, we assessed the expression of DLL4, Notch4, and ephrin B2 in transgenic mice developing hepatocarcinoma characterized by a strong remodeling of the tumor sinusoids. We also investigated the role of VEGF in the expression and biological functions of these molecules on human venous endothelial cells. In transgenic livers, we showed that DLL4, active Notch4, and ephrin B2 were gradually up-regulated within the hepatocarcinoma progression and expressed on tumor sinusoidal endothelial cells. In venous endothelial cells, we showed that VEGF up-regulates DLL4 and presenilin, and increased the activation of Notch4, leading to an up-regulation of ephrin B2 with a down-regulation of Eph B4. We also showed that the activation of Notch4 is required for VEGF-induced up-regulation of ephrin B2 and the differentiation of human venous endothelial cells in vitro. Accordingly, the disruption of Notch4 signaling by pharmacologic inhibition of presenilin or addition of soluble DLL4 inhibited the effect of VEGF on human venous endothelial cell migration and differentiation. Our study strongly suggests that a coordinated activation of DDL4/Notch4 and ephrin B2 pathways downstream of VEGF plays a key role in the abnormal remodeling of tumor vessels. (Cancer Res 2006; 66(17): 8501-10)


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The hypothesis described by Folkman three decades ago, suggesting that tumors cannot grow before the angiogenic switch, seems nevertheless more complex than thought (1, 2). Tumors do not always originate avascularly, and remodeling of preexisting vessels characterizes the tumor vasculature (3). Tumor blood vessels are leaky, irregularly shaped, dilated, lack pericytes, and form arterial-venous shunt (1, 4). Vascular endothelial growth factor (VEGF), angiopoietin, ephrin, and Notch families are critical actors for the development of a functional and specialized vascular network during embryogenesis (5, 6). The overexpression of VEGF induces enlarged blood vessels in addition to its functions on endothelial cells (7, 8). Various tumor cells or tumor stroma cells secrete angiogenic molecules, such as VEGF, which, in turn, facilitate the formation of neovessels and probably the remodeling of preexisting vessels (2–4). In the cornea, ephrin B2–positive vessel sprouts toward a VEGF pellet implant, suggesting that new vessels either arise from arteries or veins, form arterial-venous shunts, or acquire arterial markers (9). VEGF also plays a permissive role in the fate of arterial endothelial cells. In zebrafish embryos, treatment with VEGF antisense morpholino oligomers causes defects in arterial differentiation and the loss of arterial genes (10). Recently, it has been described that VEGF induces the expression of Delta-like 4 ligand (DLL4) in endothelial cells (11–13), and that VEGF treatment of mouse embryo angioblasts up-regulates the production of ephrin B2 (14).

Notch receptors and their ligands are also involved in cell fate and arterial-venous differentiation (10, 15–17). Mouse deletions of Notch1, Notch1/Notch4, presenilins, Jagged1, and Delta 1, and endothelial cell–specific knock-in of the active form of Notch4 induce embryonic lethality with vascular defect remodeling (5, 17). Recently, it has been described that the expression of constitutively active Notch4 induced abnormal vessel remodeling with arterial-venous shunts (18). In human tumor vasculature, an up-regulation of DLL4 has been described recently (12). Four Notch receptors (Notch1-Notch4) and five ligands (Jagged1 and Jagged2, and Delta1, Delta3, and Delta4) have been identified and all are single-pass transmembrane proteins (14, 19). Regulated intramembrane proteolytic processes result from the effect of Notch activation on ligand binding (14, 15). ADAM10 and presenilin induce subsequent subunit proteolysis, which releases the soluble Notch intracellular domain (14, 15). Notch intracellular domain migrates into the nucleus and associates with the CSL family of DNA-binding proteins and forms a transcriptional activator that induces downstream target genes transcription (14, 15, 19). Loss of Notch signaling induces ectopic expression of venous markers within the dorsal aorta of zebrafish embryos, whereas Notch activation induces ectopic expression of ephrin B2 in veins (16). In microvascular endothelial cells, the overexpression of constitutively active Notch4 induces ephrin B2 (20). Notch4 and its ligand Delta4 are specifically expressed in the developing endothelium of arteries (21).

Ephrin B2 is expressed on arterial endothelial cells, whereas venous endothelial cells express its main receptor, Eph B4 (22, 23). Deletion of ephrin B2 or Eph B4 results in identical phenotypes characterized by dramatic defects in vascular remodeling, maintenance of arterial-venous specialization, and vascular integrity (23). Eph receptor tyrosine kinases and their membrane-bound ephrin ligands mediate cell-to-cell communication by bidirectional signaling (22, 24, 25). Eph B4 forward signaling is required for cellular segregation and avoids cellular intermingling (26). Ephrin B2 reverse signaling can promote endothelial cell migration, adhesion, and invasion into a collagen gel and plays a role in vascular remodeling (27, 28). Unbalanced expression of the ephrin B2/Eph B4 pair within tumor vessels might be implicated in the abnormal remodeling of these tumor vessels with a loss of arterial-venous identity, as recently suggested in Kaposi sarcoma (29).

We hypothesized that endothelial cells of tumor vessels express Notch4 and DLL4, which, in turn, activate Notch4, with a subsequent increase in ephrin B2 expression. Active Notch4 and ephrin B2 reverse signaling lead to abnormal remodeling of the tumor vasculature. To support this hypothesis, we studied the expression of Notch4, Delta4, and ephrin B2 during the angiogenic process occurring in a mouse transgenic model of hepatocarcinoma characterized by strong remodeling of tumor sinusoids accompanying tumor growth (30). We showed that DLL4, active Notch4, and ephrin B2 were up-regulated in transgenic livers and expressed by the tumor sinusoidal endothelial cells. Because the normal sinusoids are of venous origin (31), these data suggest that arterial markers are acquired during tumor vessel remodeling. In cultured adult venous endothelial cells, we showed that VEGF up-regulates DLL4, ADAM10, and presenilin, and enhances the activation of Notch4 without modulation of its mRNA levels. An up-regulation of ephrin B2 with a down-regulation of EphB4 expression was observed in VEGF-treated endothelial cells. In addition, we showed that the activation of Notch4 is required for VEGF-induced up-regulation of ephrin B2 and the differentiation of human venous endothelial cells in vitro.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures. Human umbilical vein endothelial cells (HUVEC) were cultured in EBM2 supplemented with 10% FCS, 5% human serum, and 2 ng/mL fibroblast growth factor-2 (FGF-2). For cell synchronization, subconfluent HUVEC cultures were serum-deprived in EBM2 supplemented with 1% human serum for 24 hours. Synchronized HUVECs, in EBM2-0.3% bovine serum albumin (BSA) either preincubated (16 hours) or not with 10–7 mol/L of a presenilin inhibitor, L685 458, or treated or not with soluble recombinant DLL4 (rhDLL4; 1 µg/mL), were stimulated by 50 ng/mL VEGF. Cell extracts were then used for reverse transcription-PCR (RT-PCR), Western blotting, immunostaining, and functional studies. All cell culture reagents were obtained from Invitrogen (Cergy, France).

Transgenic mouse model. Transgenic C57Bl/6/ASV-B male mice developing hepatocellular carcinoma have been described elsewhere. Three major stages of the temporospatial progression of the hepatocarcinoma and angiogenic processes have been previously described (30). Livers were obtained from three transgenic mice at the main stages of hepatocarcinoma hyperplastic (4 and 8 weeks), nodular (12 weeks) and diffuse carcinoma (16 weeks), and three normal parental C57Bl/6 female mice at the same ages. RNAs, proteins, and liver sections were prepared from each liver for all experiments. Animal procedures were conducted in accordance with French government policy (Services Vétérinaires de la Santé et de la Production Animale, Ministère de l'Agriculture).

Reagents. The following antibodies were used for Western blotting and immunofluorescence staining: rabbit anti-ephrin B2 and goat anti-Delta4 (Santa Cruz Biotechnology, Tebu S.A, Le Perray en Yvelines, France), rat anti-CD31 (PharMingen Becton Dickinson, Le Pont de Claix, France), rabbit anti-Notch4 recognizing the truncated intracellular domain of int3/Notch4 (Upstate, Euromedex, Mundolsheim, France), goat anti-actin (Santa Cruz Biotechnology), donkey anti-goat IgG-peroxidase, and goat anti-rabbit IgG-peroxidase (Jackson ImmunoResearch Laboratory, West Grove, PA). Alexa Fluor 488 goat anti-rabbit, rabbit anti-goat, goat anti-rat antibodies; and Alexa Fluor 555 goat anti-rabbit, goat anti-rat, donkey anti-goat antibodies were from Interchim (Asnières, France). VEGF, FGF-2, and rhDLL4 were from R&D Systems Europe (Lille, France), and a presenilin inhibitor L685 458 was from Calbiochem (Meudon, France).

Real-time quantitative PCR. Total RNA was extracted from three transgenic and three normal livers at 4, 8, 12, and 16 weeks and from HUVECs treated for 6 hours with 50 ng/mL VEGF (four experiments) using the RNeasy Midikit and treated with the RNase-free DNase I for 15 minutes (Qiagen SA, Courtaboeuf, France). The first-strand cDNA was synthesized from 2 µg RNA from liver and 1 µg from HUVECs using a reverse transcriptase kit (Roche Diagnostics, Meylan, France) according to the instructions of the manufacturer. Quantitative RT-PCR (qRT-PCR) was done using a LightCycler system (Roche Diagnostics) according to the instructions of the manufacturer. Mouse ß-actin (liver) and eukaryotic 18S rRNA (HUVEC) were used as reference genes. The following primer sequences were used: Serial dilutions (10-fold) of one of the samples were used to generate the standard curve for each PCR. Each PCR assay was done twice using triplicate samples.


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Protein extraction and Western immunoblotting. HUVECs (107 cells) were washed with ice-cold PBS and lysed in ice-cold radioimmunoprecipitation assay (RIPA) buffer (2 mL/107 cells; ref. 32). Diced livers (0.5-2 g) were lysed in ice-cold RIPA buffer (2 mL RIPA/g liver), homogenized with 10 passes of a Dounce homogenizer, and solubilized by 30-minute incubation at 4°C with shaking. After centrifugation at 12,000 x g for 30 minutes, the supernatants were mixed in 4x Laemmli buffer, boiled, and centrifuged at 12,000 x g for 15 minutes. Protein concentrations were determined using the BCA protein reagent assay kit (Pierce, Rockford, IL). The protein samples (40 µg for HUVEC and 75 µg for mouse liver) were resolved by 7.5% and 10% SDS-PAGE under reducing conditions and transferred onto nitrocellulose membranes using 25 mmol/L Tris, 20 mmol/L glycine, and 15% methanol. The membranes were blocked by incubation in TBS containing 5% nonfat milk powder for 90 minutes at room temperature. The membranes were incubated overnight at 4°C with specific antibodies against Notch4 (1/200), DLL4 (1/200), and ephrin B2 (P-20; 1/200), with actin (1/500) used as an internal loading control. The membrane was then incubated with horseradish peroxidase–conjugated anti-rabbit (1/40,000) and anti-goat (1/30,000) IgG. Antibody binding was revealed with the ECL system (Amersham Pharmacia Biotech, Buckinghamshire, United Kingdom). Results were representative of four experiments.

Immunofluorescence staining. Liver sections were prepared as previously described (30). The primary antibody was either omitted or incubated with an excess of blocking peptide as negative control. Liver sections were incubated with rabbit anti-ephrin B2 (H-83; 1/50) or rabbit anti-Notch4 (1/20) antibodies and then incubated with an Alexa Fluor 488 goat anti-rabbit antibody (1/100). For ephrin B2 staining, the sections were permeabilized with 1% Triton X-100/PBS. For DLL4 immunostaining, the sections were incubated with goat anti-DLL4 antibody (1/20), then with an Alexa Fluor 488 rabbit anti-goat antibody (1/100). For ephrin B2/CD31 and DLL4/CD31 double immunostaining, the sections were incubated with rat anti-CD31 antibody (1/50) and then with rabbit anti-ephrin B2 antibody (1/50) or goat anti-DLL4 (1/20). This was followed by incubation with an Alexa Fluor 488 goat anti-rat antibody for CD31, and an Alexa Fluor 555 goat anti-rabbit antibody for ephrin B2 or an Alexa Fluor 555 donkey anti-goat for DLL4 (1/100). For Notch4/CD31 and Notch4/DLL4 double immunostaining, the sections were first incubated with either rat anti-CD31 (1/50) or goat anti-DLL4 (1/20) and then with a rabbit anti-Notch4 (1/20) antibody. The sections were then incubated with either an Alexa Fluor 555 goat anti-rat antibody for CD31 or a Alexa Fluor 555 donkey anti-goat antibody for DLL4 (1/100). This was followed by incubation with an Alexa Fluor 488 goat anti-rabbit antibody for Notch4 (1/100). Synchronized HUVECs, from the same cultures as used for Western blotting, were treated for 6 hours with VEGF, with or without 10–7 mol/L L685 458, fixed in acetone (90%), and permeabilized. They were then stained with anti-ephrin B2 (1/10), anti-Notch4 (1/10), or anti-DLL4 (1/20) antibodies followed by the appropriate Alexa Fluor antibodies (1/100). All immunostainings were analyzed using a standard fluorescence microscope (Zeiss, Microvision, Evry, France).

Functional assays. For [3H]thymidine incorporation assay, synchronized HUVECs were stimulated for 24 hours with 50 ng/mL VEGF, with or without a presenilin inhibitor (10–7 mol/L L685 458) or rhDLL4 (0.2-5 µg/mL), and with FGF-2 (10 ng/mL) with or without 10–7 mol/L L685 458. Methyl-[3H]thymidine (Amersham, Les Ulis, France) incorporation into DNA was measured as previously described (32). The results are expressed as means ± SD of four experiments done in triplicate as percentage of proliferation compared with control values.

Cell migration and angiogenesis assay on three-dimensional collagen gel: For wounding assays, HUVECs were cultured on gelatin-coated, 9 cm2 dishes until confluence. The monolayers were wounded using a razor blade and cell migration was induced with 50 ng/mL VEGF, with or without 10–7 mol/L L685 458 or rhDLL4 (1 µg/mL). The cultures were analyzed 8 hours later. The results were expressed as means ± SD of three experiments done in triplicate as number of migrated cells. Collagen gels (1 mg/mL, BD Biosciences, Bedford, MA) were prepared by mixing rat type I collagen solution (4 mg/mL), 10 x PBS, and 1 mol/L NaOH, in distilled water and polymerized for 30 minutes at 37°C in 24-well plates. HUVECs (106 cells/mL) in M199 were added to the collagen gels and stimulated with 50 ng/mL VEGF, with or without 10–7 mol/L L685 458 or 1 µg/mL rhDLL4, and with FGF-2 (10 ng/mL) with or without 10–7 mol/L L685 458. Tube formation was observed after 48 hours. The results (number of sprouts) were expressed as means ± SD of three experiments done in triplicate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Tumor sinusoidal endothelial cells express arterial markers. As normal liver sinusoids are of venous origin, we wondered whether the acquisition of arterial markers by tumor sinusoidal endothelial cells accompanied their remodeling. We investigated ephrin B2 in transgenic liver samples at the main stages of the tumor progression and in normal liver in age-matched mice. In normal livers, regardless of mouse age, ephrin B2 was not detected by either Western blotting (Fig. 1B ) or immunostaining (Fig. 1C) and no change in ephrin B2 mRNA levels was seen (Fig. 1A). However, ephrin B2 mRNA levels increased gradually from the nodular stage in transgenic livers (Fig. 1A). Ephrin B2 protein levels, which were barely detectable at the early stage, greatly increased from the nodular stage to the diffuse carcinoma stage (Fig. 1B). Immunostaining showed very few ephrin B2–positive cells in the hyperplastic stage, after which their numbers greatly increased from the nodular stage to the diffuse carcinoma stage (Fig. 1C). These ephrin B2–positive cells lined the nodular sinusoids perfectly. Their endothelial origin was confirmed by ephrin B2/CD31 double immunostaining (Fig. 1C).


Figure 1
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  		Figure 1. Up-regulation of ephrin B2 in transgenic livers. Temporal expression of ephrin B2 analyzed by quantitative RT-PCR (A), Western blotting (B), and immunostaining (C). RNAs, proteins, and liver sections were prepared from three transgenic livers at different stages of hepatocarcinoma: hyperplastic (4 and 8 weeks), nodular (12 weeks), and diffuse carcinoma (16 weeks), and from three normal livers at the same ages. A, the relative expression of ephrin B2 mRNA was measured by qRT-PCR. The results, or arbitrary units (A.U.), were expressed as the ratio of the amount of the RNA of interest to the amount of control RNA (mouse ß-actin). Columns, mean of three experiments; bars, SD. Mann-Whitney test was used to calculate statistics; transgenic versus normal livers: *, NS (nonsignificant); **, P < 0.03; ***, P < 0.001; ****, P < 0.0003. B, representative Western blots showed a temporal expression of ephrin B2 in transgenic livers, whereas ephrin B2 was not detectable in normal livers. Equal loading of proteins was assessed by imaging Ponceau S staining transfer membrane, and quantification of total protein content in each lane was determined by densitometric analysis. C, no ephrin B2 staining was detected in normal livers regardless of mouse age. In transgenic livers, ephrin B2–positive sinusoidal endothelial cells were detected from an early stage (8 weeks). The number of these cells increased at the nodular and diffuse stages, and completely lined the sinusoids (arrows). Original magnification, x40. Ephrin B2/CD31 double immunostaining in transgenic livers at the diffuse stage: ephrin B2 (red); CD31 (green). Most CD31-positive endothelial cells expressed ephrin B2 (arrows). Original magnification, x40.

 
Because Notch4 acts upstream of ephrin B2, we therefore investigated Notch4 and DLL4 in the same liver samples. Both Notch4 and DLL4 mRNA levels were constant in normal livers (Fig. 2A ). In transgenic livers, Notch4 and DLL4 mRNAs increased significantly from the nodular to the diffuse carcinoma stage compared with normal livers (Fig. 2A). At the hyperplastic stage, the truncated, active form of Notch4 protein was barely detected by Western blotting, and its level greatly increased from the nodular stage (Fig. 2B). Under these experimental conditions, the full length of Notch4 protein was not detectable. From the hyperplastic to the diffuse carcinoma stage, this increase was associated with a lower increase in DLL4 protein levels, whereas Notch4 and DLL4 proteins were not detected by Western blotting in normal livers (Fig. 2B). Notch4 immunostaining was not seen in control livers (Fig. 2C) or in transgenic livers at the early hyperplastic stage (Fig. 2C). However, strong Notch4 staining in cells lining nodular sinusoids was seen at the nodular stage and was increased at the diffuse carcinoma stage (Fig. 2C). We confirmed the endothelial origin of Notch4-positive cells by Notch4/CD31 double immunostaining (Fig. 2C). A similar pattern of DLL4 staining was seen in sinusoidal endothelial cells in transgenic livers as confirmed by DLL4/CD31 double immunostaining (Fig. 2C), whereas no DLL4 staining was observed in normal livers (data not shown). Notch4/DLL4 double immunostaining showed a coexpression of these proteins in sinusoidal endothelial cells in transgenic livers (Fig. 2C). This suggested that the presence of both receptor and ligand on adjacent endothelial cells leads to in situ activation of Notch4.


Figure 2
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  		Figure 2. Notch4 and Delta4 are up-regulated in transgenic livers. qRT-PCR analysis of Notch4 and DLL4 (A), Western blot analysis of Notch4 and DLL4 (B), and immunostaining (C). RNAs, proteins, and liver sections were prepared from the same liver samples as mentioned in Fig. 1. A, results and statistical analysis of qRT-PCR were described in Fig. 1. For Notch4 mRNA: *, P < 0.003; **, NS; ***, P < 0.003; ****, P < 0.0002. For DLL4 mRNA: *, NS; **, NS; ***, P < 0.001; ****, P < 0.0002. B, representative Western blots showed a temporal expression of Notch4 and DLL4 in transgenic livers, whereas they were not detectable in normal livers. Equal loading of proteins was assessed as described in Fig. 1. C, Notch4 staining was not detected in sinusoidal endothelial cells from normal livers regardless of mouse age or from transgenic livers at the hyperplastic stage (8 weeks). Notch4 was expressed by endothelial cells lining sinusoids (arrows) from the nodular stage (12 weeks), with increased expression at the diffuse stage (16 weeks) in transgenic livers. The sinusoidal network was dilated and fused. Original magnification, x40. Notch4/CD31 double immunostaining at the diffuse stage of hepatocarcinoma: Notch4 (green); CD31 (red). Most of the CD31-positive sinusoidal endothelial cells expressed Notch4 (arrows and inset). Original magnification, x40; inset, x100. DLL4 immunostaining was not detected in transgenic livers at early stages. DLL4-positive endothelial cells lining sinusoids were detected from the nodular stage (12 weeks), and this staining (arrows) increased in the diffuse stage (16 weeks). Original magnification, x40. DLL4/CD31 double immunostaining at the diffuse stage of hepatocarcinoma: DLL4 (green); CD31 (red). Most of the CD31-positive sinusoidal endothelial cells expressed DLL4 (arrows and inset). Original magnification, x40; inset, x100. Notch4/DLL4 double immunostaining in transgenic livers at the diffuse stage: Notch4 (green); DLL4 (red). DLL4 and Notch4 were expressed together in tumor sinusoidal endothelial cells (arrows and inset). Original magnification, x40; inset, x100.

 
HUVECs express arterial markers on VEGF treatment. To determine whether VEGF promotes arterial phenotypic switching, we stimulated HUVECs with VEGF, and tested for EphB4 and ephrin B2 expression. Ephrin B2 mRNA levels increased 9-fold, whereas EphB4 mRNA levels decreased 2-fold (Fig. 3A ). We also studied the expression of Notch4 as its signaling acts upstream of ephrin B2 in microvascular endothelial cells. Notch4 mRNA levels did not differ significantly in control and VEGF-treated HUVECs (Fig. 3A). Western blot analysis of the same HUVEC cultures showed increased levels (x3) after 1 hour of the truncated form of Notch4, which reached a plateau after 2 hours (Fig. 3B). Immunostaining showed a nuclear localization of Notch4 on VEGF treatment, whereas this was not seen for control HUVECs (Fig. 3C). Upon VEGF treatment, DLL4 mRNA levels increased 3-fold (Fig. 3A). This up-regulation of DLL4 was confirmed by Western blotting (Fig. 3D). Immunostaining showed a membrane localization of DLL4, whereas this was not seen for control HUVECs (Fig. 3C). Taken together, these results were in favor of an activation of Notch4 on ligand binding. In the same HUVEC cultures, the increase in ephrin B2 levels was delayed, reaching a plateau from 6 to 16 hours (Fig. 3B). VEGF increased the number of ephrin B2–positive cells (Fig. 3C).


Figure 3
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  		Figure 3. VEGF induces an arterial phenotype in HUVECs. A, qRT-PCR for ephrin B2, Notch4, DLL4, and Eph B4 in HUVECs stimulated or not with VEGF (50 ng/mL) for 6 hours. Columns, mean of four experiments expressed as the ratio of the amount of the RNA of interest to the amount of 18S rRNA; bars, SD. Mann-Whitney test was used to calculate statistics. VEGF-treated HUVECs versus unstimulated HUVECs: *, P < 0.0001; **, NS; ***, P < 0.005; ****, P < 0.01. B, detection of Notch4 and ephrin B2 by Western blotting and time course of Notch4 and ephrin B2 expression. The signal corresponds to the active form of Notch4. Results were representative of four experiments. Points, mean; bars, SD. Significant increase of Notch4 levels was observed 1 hour after VEGF treatment (*, P < 0.05). The significant increase in ephrin B2 levels was delayed, reaching a plateau from 6 hours (**, P < 0.05). For each experiment, the intensity of Notch4 and ephrin B2 bands was normalized to that of nonstimulated HUVECs. C, under a 6-hour VEGF treatment, a nuclear localization of Notch4 was observed, whereas DLL4 staining was localized in the membrane, with an increase of ephrin B2–positive HUVECs. Magnification, x40. D, an up-regulation of DLL4 expression was depicted by Western blotting in VEGF-treated HUVECs for 6 hours.

 
VEGF-induced ephrin B2 in venous endothelial cells is related to DLL4/Notch4 signaling. We first investigated the effect of VEGF on the two enzymes involved in Notch4 activation on ligand binding. We showed that VEGF up-regulated both presenilin (10-fold) and ADAM10 mRNA (13-fold) levels (Fig. 4A ). We therefore wondered whether the inhibition of presenilin impaired Notch4 activation and subsequently ephrin B2 expression. To address this question, HUVECs were preincubated (16 hours) or not with L685 458 and then stimulated by VEGF for 6 hours. The formation of the VEGF-induced truncated form of Notch4 was greatly inhibited by L685 458 (Fig. 4C). We showed that L685 458 reduced the number of ephrin B2–positive cells in VEGF-treated HUVECs (Fig. 4B). We investigated the effect of rhDLL4 on VEGF-induced Notch4 cleavage in HUVECs. We also showed that rhDLL4 significantly reduced the expression of cleaved Notch4 (Fig. 4D) and inhibited Notch4 nuclear staining (Fig. 4D).


Figure 4
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  		Figure 4. VEGF-induced ephrin B2 in HUVECs is related to DLL4/Notch4 signaling. A, qRT-PCR for Adam10 and presenilin in HUVECs stimulated or not with VEGF (50 ng/mL) for 6 hours. Results of four experiments were expressed as the ratio of the amount of the RNA of interest to the amount of 18S rRNA. Columns, mean; bars, SD. VEGF up-regulates ADAM 10 and presenilin in HUVECs. Student's t test was used to calculate statistics. VEGF-treated HUVECs versus unstimulated HUVECs: *, P < 0.001; **, P < 0.005. B, the presenilin inhibitor (L685 458) greatly reduced the number of ephrin B2–positive cells in VEGF-treated HUVECs for 6 hours. Magnification, x40. C, effect of L685 458 on Notch4 expression by Western blot in HUVECs stimulated with VEGF for 6 hours, representative experiment, and quantitative analysis of Notch4. Results were expressed as the ratio of Notch4 to actin absorbance (O.D.) values obtained for each condition. Columns, mean; bars, SD. Student's t test was used to calculate statistics: *, VEGF versus control, P < 0.01, **, VEGF + L685 458 versus VEGF, P < 0.02; ***, VEGF + L685 458 versus L685 458, NS. D, Western blotting analysis of cleaved Notch4 in VEGF-treated HUVECs for 6 hours with and without rhDLL4. Columns, mean of three experiments; bars, SD. *, VEGF versus control, P < 0.009; **, VEGF + DLL4 versus VEGF, P < 0.006. RhDLL4 reduced the Notch4 nuclear labeling in VEGF-treated HUVECs. Magnification, x40.

 
VEGF-induced migration and differentiation of venous endothelial cells depend on DLL4/Notch4 signaling. We further investigated the role of Notch4 signaling in venous endothelial cell responses to VEGF. We evaluated whether an inhibitor of presenilin (L685 458) or inhibition of Notch4-DLL4 binding impaired venous endothelial cell functions on VEGF treatment. HUVECs migrated in the presence of VEGF in a lateral migration assay (Fig. 5A ). L685 458 and rhDLL4 reduced HUVEC migration upon VEGF treatment by 63% and 65%, respectively, whereas L685 458 had no effect on FGF-2-treated HUVECs (Fig. 5A).


Figure 5
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  		Figure 5. Inhibition of DLL4/Notch4 signaling in VEGF-treated HUVECs reduces migration and differentiation. A, wound healing assay of HUVECs induced by VEGF (50 ng/mL) or FGF-2 (10 ng/mL), with or without L685 458 (10–7 mol/L) or rhDLL4 (1 µg/mL), and analyzed 8 hours later. Magnification, x20. The results of counted migrated cells were representative of three experiments each done in triplicate. Columns, mean; bars, SD. Student's t test was used to calculate statistics: *, VEGF + L685 458 versus VEGF, P < 0.01; **, VEGF + rhDLL4 versus VEGF, P < 0.01; ***, FGF-2 + L685 458 versus FGF-2, NS. B, angiogenesis assay on three-dimensional collagen gels: HUVECs were seeded onto collagen gels and stimulated with VEGF (50 ng/mL) or FGF-2 (10 ng/mL), with or without L685 458 (10–7 mol/L) or rhDLL4 (1 µg/mL). The pictures showed the HUVEC network after 24 hours of treatment. Magnification, x20. Results were representative of three experiments each done in triplicate. Columns, mean of sprout numbers; bars, SD. Either L685 458 or DLL4 induced a significant inhibition of HUVEC sprouting (*, P < 0.000005; **, P < 0.00005, respectively). C, left, L685 458 did not significantly inhibit HUVEC proliferation induced either by VEGF or FGF-2. The control for HUVEC proliferation in the presence of L685 458 is medium + DMSO (1/10,000). Results were expressed as percentage of control values. Columns, mean of four experiments done in triplicate; bars, SD. *, VEGF + L685 458 versus VEGF, NS; **, FGF-2 + L685 458 versus FGF-2, NS. Right, dose response of rhDLL4 (0.2-5 µg/mL) on HUVECs proliferation induced either by VEGF or FGF-2. Results were expressed as percentage of control values. Columns, mean of three experiments done in triplicate; bars, SD. *, VEGF + rhDLL4 versus VEGF, NS; **, FGF-2 + rhDLL4 versus FGF-2, NS.

 
VEGF and FGF-2 induced differentiation of HUVECs into capillary-like structures in the three-dimensional collagen gel with numerous, highly-branched sprouting networks (Fig. 5B). L685 458 reduced by 88% the amount of sprouting on VEGF-treated HUVECs, whereas it had no effect on FGF-treated HUVECs (Fig. 5B). rhDLL4 reduced by 71% the HUVECs network under VEGF treatment (Fig. 5B).

As expected, VEGF and FGF-2 caused an increase of HUVEC proliferation (Fig. 5C). Both L685 458 (Fig. 5C) and rhDLL4 (Fig. 5C) did not significantly modify HUVEC proliferation stimulated by VEGF or FGF-2.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Tumor angiogenesis is not only characterized by the appearance of new vessel sprouts but also by structurally and functionally abnormal vessels (3, 4). Despite advances in the field of embryogenesis, molecular pathways acting downstream of VEGF in controlling the remodeling of the tumor vessels are still poorly documented. Many tumor vessels depend on VEGF for survival and VEGF induced vascular malformations (33). Genetic and molecular studies suggest that the VEGF, Notch, and ephrin families play a key role in vascular development during embryogenesis (5, 19, 23, 24). A recent study have reported that DLL4 was expressed in renal cancer (12). Few studies have shown that ephrin B2 marks remodeled vessels at sites of adult neovascularization (9, 29, 34). We investigated whether the VEGF-Notch4-ephrin B2 cascade could participate in the vessel remodeling observed in tumor angiogenesis. We used a transgenic hepatocarcinoma model that we have already characterized as a strong remodeling of tumor sinusoids accompanying the angiogenic process (30). VEGF is up-regulated in this model (30), as in human hepatocarcinoma (35). Furthermore, the liver sinusoidal vessels are of venous origin and the liver sinusoidal endothelial cells differ morphologically and functionally from capillary endothelial cells of other organs in physiologic conditions (31). Liver sinusoids are appropriate vessels for the study of the acquisition of arterial markers during vascular remodeling. We showed a temporospatial expression of DLL4, active Notch4, and ephrin B2 during remodeling of the tumor sinusoids. Ephrin B2, active Notch4, and DLL4 were restricted to the tumor sinusoidal endothelial cells and were present at the early stages of hepatocarcinoma. The presence of Notch4 and DLL4 on the sinusoidal endothelial cells may activate Notch4 in situ. The observed accumulation of the truncated form of Notch4 in transgenic livers is consistent with such activation. VEGF and DLL4 are the only genes whose the haploinsufficiency leads to embryonic lethality due to vascular malformations (36). Recently, an up-regulation of DLL4 correlated with VEGF expression has been shown in clear renal cell carcinoma (12). The expression of constitutively active Notch4, int3 in the adult mouse endothelium, using the tetracycline-repressible system, induced lethality with tortuous, large vessels, and arterial-venous shunts (18). An arteriolization accompanied these int3-mediated vascular defects with ectopic venous expression of ephrin B2 and up-regulation of DLL4 (18). Instead of VEGF, known to cause blood vessel dilation, the observed accumulation of the truncated active form of Notch4 in transgenic livers probably induced, in part, the abnormal tumor sinusoidal remodeling. Ephrin B2 reverse signaling also played a role in vascular remodeling (24, 28). EphB4 induces tumor growth by stimulating angiogenesis through ephrin B2 (27). Ephrin B2, but not EphB4 transcripts, were detected in Kaposi sarcoma, suggesting that the Kaposi tumor cells are of endothelial origin with an arterial phenotype (29). Consequently, the imbalance between Eph B4 and ephrin B2 observed in tumor sinusoidal endothelial cells might promote also a cue for the fusion of liver sinusoids in this model.

We further investigated whether VEGF induces the acquisition of these arterial markers in adult venous endothelial cells. We showed that VEGF induced the expression of DLL4 as previously described (12). A rapid accumulation of the truncated form of Notch4, without significant change in Notch4 mRNA levels, was observed. Notch4 staining was seen in the nucleus of VEGF-treated HUVECs, suggesting that Notch4 was activated. It has been previously reported that VEGF induced Notch1 and DLL4 mRNAs in arterial endothelial cells but did not modify Notch4 mRNA level in HUVECs (11). We confirmed that VEGF does not modulate Notch4 mRNA level in HUVECs, but, interestingly, we showed that VEGF regulates Notch4 activation by acting at a posttranscriptional level. We concluded that VEGF up-regulates DLL4 in endothelial cells, which leads to the activation of Notch4 on neighboring endothelial cells. ADAM10 and presenilin have been implicated in this proteolysis of Notch receptors (14, 15, 37). The nuclear localization of Notch4 suggests that the enzymes cleaving Notch4 are activated and, in fact, we found that ADAM10 and presenilin mRNA levels are up-regulated by VEGF. In these venous endothelial cell cultures, ephrin B2 mRNA level increased 9-fold, whereas EphB4 mRNA level decreased 2-fold. Western blot analysis confirmed the increase in ephrin B2 level and showed that there was a delay between Notch4 cleavage and the increase in ephrin B2 level. This suggests that ephrin B2 up-regulation occurs downstream of Notch4 cleavage. Immunofluorescence studies also showed that control HUVECs contained few ephrin B2–positive cells and that VEGF greatly increased their number. In agreement with our results, the down-regulation of DLL4 expression led to a decrease of the expression of ephrin B2 (12). It would therefore appear that VEGF switched the HUVEC phenotype from venous to arterial. We further wondered whether disruption of Notch4 signaling could modify ephrin B2 expression and venous endothelial cell responses upon VEGF treatment. Presenilin inhibitors are known to inhibit the Notch pathway (38, 39). We showed that the pharmacologic inhibition of presenilin strongly reduced the accumulation of the truncated form of Notch4. This subsequently reduced ephrin B2 expression in VEGF-treated HUVECs. In our study, L685 458 had no effect on HUVECs proliferation induced by VEGF or FGF-2. In contrast, the presenilin inhibitor strongly reduced the remodeling activity of VEGF, mimicked in vitro by the migration and the three-dimensional collagen gel assays. In favor of a VEGF specificity, L685 458 had no effect on the HUVECs network and migration in response to FGF-2. Constitutive activation of Notch signaling inhibited human iliac artery endothelial cell proliferation but increased the endothelial cell survival and network formation in Matrigel or in three-dimensional collagen gel, whereas blocking of Notch signaling inhibited network formation in three-dimensional collagen gel (11). The activation of Notch signaling by Jagged1 inhibited HUVEC and human aortic endothelial cell proliferation, whereas Jagged1 induced network formation in vitro (40). Recently, it has been shown that Jagged1-expressing tumor cells triggered capillary-like network formation through Notch signaling in endothelial cells (41), and that down-regulation of DLL4 inhibited endothelial cell proliferation, migration, and network formation. This network was inhibited by presenilin inhibitor (41). In vitro, the effect of Notch signaling on endothelial cell functions seemed to depend on the endothelial cell type and was likely to involve differentiation rather than proliferation. In our study, HUVEC migration and differentiation induced by VEGF was reduced by presenilin inhibitor, whereas proliferation was not. {gamma}-Secretase inhibitors are not specific of presenilin and can target other proteins (42), but studies have shown their role in angiogenesis in vitro and in vivo (43–45). The antiangiogenic effect of the {gamma}-secretase inhibitors probably involves different pathways, including Notch signaling.

As Notch signaling requires cell-to-cell contact of the membrane-associated Notch and ligand (14, 15, 19), we then wondered whether competitive inhibition of VEGF-induced DLL4-Notch4 binding impaired Notch4-mediated functional effects on venous endothelial cells. We showed that a soluble form of DLL4 inhibited migration and network formation of VEGF-treated HUVECs without any effect on HUVEC growth. Recently, a down-regulation of VEGF receptor-2 and Neuropilin1 with reduced endothelial cells functions to VEGF was described in HUVECs retrovirally transduced with DLL4 (13). The presenilin inhibitor L685 458 reconstituted VEGF receptor-2 and endothelial cells responses to VEGF in DLL4-transduced endothelial cells, supporting the role of Notch signaling (13). The transfection of constitutively activated Notch1 or Notch4 in endothelial cells down-regulates VEGFR-2 with a decrease of endothelial cell proliferation in response to VEGF but not to FGF (46). Although these studies used endothelial cells overexpressing DLL4 or Notch, they point to the regulatory role of Notch signaling on endothelial cells functions to VEGF. A cross-talk between tumor cells expressing Jagged1 leading to the activation of Notch in endothelial cells and tumor angiogenesis has been described (41, 47). We propose a different putative mechanism of tumor angiogenesis and remodeling. In response to growth factors, such as VEGF, secreted by the tumor cells, endothelial cells sustain high levels of bioavailable DLL4 leading to Notch4 activation and the up-regulation of ephrin B2 on neighboring endothelial cells (Fig. 6 ). In addition, VEGF up-regulates ADAM10 and presenilin, which sustains Notch4 activation.


Figure 6
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  		Figure 6. VEGF contributes positively to the Notch4 pathway and the up-regulation of ephrin B2 in endothelial cells. Tumor cells secrete growth factors such as VEGF. VEGF increases the expression of DLL4, which binds to the Notch4 receptor on adjacent endothelial cells (transinteraction), leading to an increase of Notch4 activation. VEGF up-regulates presenilin and ADAM10, which sustain Notch4 activation. Ligand binding induces the release of a free intracellular domain of Notch4 (NICD). The truncated active form of Notch4 (NICD) translocates to the nucleus and activates the expression of ephrin B2. Notch4/DLL4 pathway and ephrin B2 reverse signaling induce abnormal vessel remodeling.

 
Normalization of tumor vasculature may be valuable for antiangiogenic therapy. The inhibition of VEGF signaling tends to normalize tumor vasculature (4). Therefore, disrupting the VEGF-DLL4/Notch4-ephrin B2 cascade downstream of VEGF may be safer than inhibiting VEGF itself and may also induce vessel maturation.


   Acknowledgments
 
Grant support: Association de la Recherche pour le Cancer (CL 3124), the Ligue Nationale Contre le Cancer, and l'Agence Nationale de la Recherche.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank Service de gynécologie-obstétrique de l'hôpital Lariboisière.


   Footnotes
 
Note: P. Hainaud and J-O. Contrerès contributed equally to this work.

Received 11/29/05. Revised 6/15/06. Accepted 6/27/06.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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