Correspondence to Ilaria Cascone: ilaria.cascone{at}curie.fr
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I. Cascone's present address is Institut Curie, INSERM U528, rue d'Ulm 26, 75248 Paris Cedex 05, France.
Abbreviations used in this paper: Ab, antibody; anti-CBP, anti-fibronectin cell binding peptide mAb; CAM, chick chorioallantoic membrane; EC, endothelial cell; ECM, extracellular matrix; MTT, [(3-4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium]; PAE, porcine aortic endothelial; PI3-K, phosphatidylinositol-3-kinase; VEGF, vascular endothelial growth factor. VEGFR, vascular endothelial growth factor receptor.
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Introduction |
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Ang-1 is the ligand of the endothelial tyrosine kinase receptor, Tie2 (Davis et al., 1996). Mice lacking Ang-1 die during embryo development (E12.5) showing a poorly remodeled and mature vasculature with defects in EC adhesion and spreading to the underlying ECM (Suri et al., 1996). The role of Ang-1 in adult angiogenesis is controversial. Several investigators have shown that Ang-1 acts as proangiogenic factor, whereas others have demonstrated the opposite (Suri et al., 1998; Chae et al., 2000; Hangai et al., 2001; Hawighorst et al., 2002; Shim et al., 2002; Uemura et al., 2002; Stoeltzing et al., 2003). However, in vitro Ang-1 promotes a proangiogenic program in ECs characterized by expression of metalloproteases and plasmin, and induction of morphogenesis, motility, and survival (Koblizek et al., 1998; Papapetropoulos et al., 1999; Cascone et al., 2003a; Das et al., 2003). It recently was demonstrated that Ang-1 promotes cell adhesion (Arai et al., 2004; Lemieux et al., 2005), and that this process is mediated by 5-integrin in ECs (Carlson et al., 2001). Moreover, the finding that Ang-1 can bind ECM extracts from carcinoma cells (Xu and Yu, 2001) has offered new insights to understand the role of Ang-1 in modulating the angiogenic microenvironment.
Cell adhesion is mediated by integrin heterodimers (Giancotti and Ruoslahti, 1999). Cross-talks between integrins and growth factor receptors were shown to coordinate biologic processes through the regulation of downstream and inside-out signaling pathways (Schneller et al., 1997; Soldi et al., 1999; Byzova et al., 2000; Sieg et al., 2000; Baron et al., 2002; Lee and Juliano, 2002). Tyrosine kinase receptors and integrins share many downstream effectors. In particular, activated Tie2 recruits p85, phosphorylates FAK, and modulates Rho GTPases (Kontos et al., 1998; Jones et al., 2001; Cascone et al., 2003a), which also participate in outside-in integrin signaling (Hood and Cheresh, 2002).
Integrins have crucial roles in angiogenesis (Hodivala-Dilke et al., 2003) and allow vascular cells to adapt their adhesive machinery to the so-called "provisional" ECM components, like fibronectin, collagen, and vitronectin, that are exposed by basement degradation around sprouting vessels (Kalluri, 2003). Integrins vß3,
vß5,
2ß1, and
5ß1 are up-regulated in newly formed blood vessels (Max et al., 1997; Kim et al., 2000b), and
vß3 and
vß5 antagonists inhibit in vitro and in vivo angiogenesis (Brooks et al., 1995; Drake et al., 1995; Hammes et al., 1996).
2-blocking antibodies (Abs) inhibit vascular endothelial growth factor (VEGF)-Ainduced angiogenesis (Senger et al., 1997). Vascular defects are described in
5-null embryoid bodies and teratocarcinomas (Taverna and Hynes, 2001; Francis et al., 2002); antagonists of the central cell-binding domain of fibronectin also inhibit angiogenesis (Kim et al., 2000b). Integrins can exist in different functional states that regulate their biologic functions (Hynes, 2002). In vivo integrin activity depends on the extracellular environment; it has been shown that modulation of ECM concentration and patterning leads to different cell responses ranging from apoptosis to growth and differentiation (Dike et al., 1999).
Here, we hypothesize that Ang-1/Tie2 could mediate different biologic effects under the influence of integrin activity. We demonstrate that Tie2 and 5ß1 form a constitutive and specific complex, and that Ang-1/Tie2 system is sensitized by
5ß1 engagement to fibronectin. Furthermore, we show that
5ß1 function is essential to mediate in vivo Ang-1dependent angiogenesis in a chick chorioallantoic membrane (CAM) assay.
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Results |
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We analyzed the influence of the integrin function on Ang-1mediated EC survival; plating ECs on ECM largely involved in angiogenesis, such as fibronectin, collagen I, and fibrinogen, ligands for 5ß1,
2ß1 and
1ß1, and
vß3 integrins, respectively. ECs were starved and kept for 24 h in 0.5% FCS in the presence of increasing concentrations of Ang-1 (Fig. S1 a; available at http://www.jcb.org/cgi/content/full/jcb.200507082/DC1). In cells plated on collagen I or fibrinogen, the survival effect of Ang-1 reached relevant values at 200 ng/ml, and the plateau condition at 500 ng/ml. When fibronectin was used as substrate, Ang-1 was effective at 100 ng/ml and attained the plateau at 200 ng/ml. These results demonstrated a shift of the doseresponse of EC survival on fibronectin versus lower Ang-1 concentration. This effect of fibronectin was not observed when ECs were stimulated with VEGF-A165 (Fig. S1 b).
Because the doseresponse effect of Ang-1 on EC survival was dependent on ECM components (Fig. S1 a), we analyzed the effect of specific integrin engagement on Tie2 activation. To verify if ECM could modify Tie2 phosphorylation doseresponse, we first established Tie2 phosphorylation in ECs plated on native ECMs (Fig. 1, a and b). Next, on the basis of the curve in Fig. 1 b, we stimulated ECs plated on fibronectin, collagen I, or fibrinogen with an ineffective concentration of Ang-1 (20 ng/ml), and with a higher dose (100 ng/ml) that triggered a 3.15-fold increase of Tie2 phosphorylation (Fig. 1 c). ECs plated on collagen I or fibrinogen showed a Tie2 phosphorylation response (Fig. 1, c and d) similar to ECM native conditions (Fig. 1 b). On fibronectin, ECs underwent a marked Tie2 phosphorylation with 20 ng/ml of Ang-1 (4.4-fold increase), whereas 100 ng/ml was less effective (2.2-fold increase; Fig. 1, c and d). As control, Fig. 1 g shows that the effect of a concentration of VEGF-A165 that induces a negligible phosphorylation of VEGF receptor (VEGFR)-2 was not affected by ECM proteins, including fibronectin.
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Tie2 interacts selectively and constitutively with 5ß1
We investigated whether Tie2 receptor could interact physically with integrins, in particular with 5ß1, the main fibronectin receptor. Lysates from ECs cultured on native ECM were immunoprecipitated with anti-
5ß1 (AB1950) or anti-ß1A (Fig. 2 a), anti-
2ß1 (BHA2.1) or anti-
2ß1 (JBS2) (Fig. 2 b), anti-
vß3 (LM609), or anti-
vß3 (25E11) (Fig. 2 c) Abs and blotted with anti-Tie2 Ab. Tie2 coimmunoprecipitated with
5ß1, but not with
2ß1 or
vß3 integrins. VEGFR-2 was not detected in
5ß1 immunoprecipitates (Fig. 2 a). To confirm further the specificity of the association between Tie2 and
5ß1,
5ß1 immunoprecipitates were re-immunoprecipitated with anti-Tie2 and blotted with anti-Tie2 Ab (Fig. 2 d). However, Tie2/
5ß1 association was not detectable by blotting Tie2 immunoprecipitation with anti-
5ß1 (unpublished data), as it has been described for other integrin/tyrosine kinase receptor complexes (Baron et al., 2002; Woodard et al., 1998). To gain further insights about the specificity of Tie2/
5ß1 interaction, CHO lacking (CHO B2) or expressing (CHO B2a27)
5 subunit were transfected transiently with Tie2, and immunoprecipitated with an anti-ß1 Ab (Fig. 2 e). Tie2 coimmunoprecipitated with ß1 only in CHO B2a27 cells (Fig. 2 e). Thus, Tie2 interacts selectively and constitutively with
5ß1 and not with other ß1 heterodimers. Then, we labeled, by biotinylation, cell surfaces of CHO B2 and CHO B2a27 expressing Tie2, and we re-immunoprecipitated Tie2 from ß1 immunoprecipitates as above. Biotinylated Tie2 was present only in ß1 immune complexes that were isolated from CHO B2a27 (Fig. 2 f). This strengthened the specificity of the Tie2/
5ß1 interaction and demonstrating that it occurred at the plasma membrane.
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In ECs plated on native ECM, we observed that Tie2 is phosphorylated after 5 min and is active until 1 h of Ang-1 stimulation (unpublished data). To demonstrate that Tie2 sensitization to low Ang-1 concentration was 5ß1-dependent, we stimulated CHO B2 and CHO B2a27 expressing Tie2 or VEGFR-2 for up to 60 min (Fig. 2, gl). Ang-1 promoted a higher degree of Tie2 phosphorylation in CHO B2a27 compared with CHO B2 (Fig. 2, g and h). Furthermore, in CHO cells lacking
5, Tie2 phosphorylation declined after 15 min (Fig. 2, g and h). The phosphorylation response of VEGFR-2 to its ligand (Fig. 2, i and l) in the two cell lines did not exhibit the clear-cut differences that were observed for Tie2; this suggested that
5ß1 specifically enhances and prolongs Ang-1induced Tie2 phosphorylation.
The Tie2/5ß1 complex is induced by fibronectin and activated by Ang-1
Then we investigated the regulation of Tie2/5ß1 interaction in ECs (Fig. 3). When ECs were plated on fibronectin, the amount of Tie2 coimmunoprecipitated with
5ß1 integrin was remarkably higher than on collagen I or fibrinogen (Fig. 3, a and b). The ability of fibronectin to increase this interaction was dose-dependent (Fig. 3 c), whereas Ang-1 stimulation did not modify the amount of receptor that coimmunoprecipitated with
5ß1 integrin on fibronectin, collagen I, or fibrinogen (Fig. 3 d and data not depicted). To analyze the signaling activity of Tie2/
5ß1 interaction, we assessed the recruitment of the p85 subunit of PI3-K and FAK to the complex. p85 is an effector of activated Tie2 (Kontos et al., 1998), whereas FAK localizes to integrin clusters and participates in the integrin signaling (Hood and Cheresh, 2002). Tie2 association with fibronectin-activated
5ß1 was not accompanied by the recruitment of FAK and p85 in the
5ß1 immunoprecipitates (Fig. 3 c). EC stimulation by Ang-1 did not modify Tie2 association with
5ß1, but did increase FAK and p85 recruitment to the complex (Fig. 3 d). Thus,
5ß1 occupancy regulates the stoichiometry of Tie2/
5ß1 association, whereas Ang-1 triggers intracellular signals downstream of Tie2/
5ß1 complexes.
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Ang-1 selectively enhances EC motility on fibronectin
We demonstrated previously that Ang-1 promotes EC chemokinesis by increasing their basal locomotion (Cascone et al., 2003a). We analyzed EC motility on fibronectin, collagen I, and fibrinogen by time-lapse videomicroscopy experiments in the presence or absence of Ang-1. Ang-1 (50 ng/ml) greatly increased the basal speed of ECs plated on fibronectin, whereas its effect was less on cells plated on collagen I and fibrinogen (Fig. 4 a). The net path covered and the migration persistence also were greater on fibronectin (Fig. 4, b and c). In contrast, Ang-1 did not modify the net path covered and had a slight effect on cell persistence on collagen I or fibrinogen (Fig. 4, b and c). The results did not change under increasing Ang-1 concentrations, independently from the type of substrate (unpublished data). Fig. 4 d shows the tracks of single ECs; this clearly indicates that Ang-1 stimulated a persistent motility of EC on fibronectin, whereas on collagen I and fibrinogen, cells moved randomly around the starting point. As expected (Rousseau et al., 2002; Serini et al., 2003a) VEGF-A165 was able to increase all chemokinetic parameters studied, but it did not show gains of function on fibronectin compared with the other ECM components (Fig. 4, ac); this supported the specificity of Ang-1/Tie2 signaling.
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To support the chemokinetic results further, we studied Ang-1 activity in haptotaxis (Fig. S3 a; available at http://www.jcb.org/cgi/content/full/jcb.200507082/DC1). Ang-1 increased cell migration toward all the substrates tested in a dose-dependent manner. However, the doseresponse effect of Ang-1 toward fibronectin was shifted to the left and reached the plateau at 50 ng/ml. Ang-1dependent haptotaxis activity toward collagen I or fibrinogen was less, appeared at 50 ng/ml, and attained the plateau at 100 ng/ml. VEGF-A165 did not showed any selective effect on EC motility toward fibronectin as compared with collagen I and fibrinogen (Fig. S3 b).
Ang-1 enhances integrin-mediated EC adhesion
Growth factor receptors modulate integrin function through PI3-Kdependent inside-out signaling to mediate enhanced biologic effects, such as cell motility, survival, and invasion (Guilherme et al., 1998; Byzova et al., 2000; Baron et al., 2002). Therefore, we verified if the Ang-1enhanced effect on fibronectin involved selective 5ß1 inside-out activation. Then we performed adhesion assays of ECs to fibronectin, collagen I, fibrinogen (Fig. 5), and vitronectin (not depicted) in the presence or absence of Ang-1. Surprisingly, adhesion assays with different concentrations of Ang-1 and ECM proteins showed that Ang-1 increased EC adhesion to a similar extent, independently from the engaged integrins. The highest increase was observed at 50 ng/ml of Ang-1 in ECs adhering on 5 µg/ml of fibronectin or collagen I and 20 µg/ml of fibrinogen (Fig. 5, a, c, and e). Function blocking anti-
5ß1 (Fig. 5 b), anti-
2ß1 (Fig. 5 d), and anti-
vß3 (Fig. 5 f) mAbs impaired Ang-1increased EC adhesion to fibronectin, collagen I, and fibrinogen, respectively; this demonstrated that Ang-1mediated adhesion was integrin-dependent. Because
vß3 promotes RGD-mediated cell adhesion (Arnaout et al., 2002), anti-
vß3 mAb partially inhibited basal EC spreading, but did not block Ang-1mediated adhesion to fibronectin (Fig. 5 b and not depicted). The PI3-Kspecific inhibitor, LY294992, affected Ang-1 proadhesive activity on all ECM ligands (Fig. 5, b, d, and f); this indicated that PI3-K plays a role in this Tie2-dependent inside-out signaling. The role of Tie2 activation in EC adhesion induced by soluble Ang-1 also was investigated in porcine aortic ECs (PAE cells) that were transfected with Tie2 (PAE-Tie2) or with the mutant Tie2-K854R (PAE-Tie2-K854R), which lacks the catalytic activity (Audero et al., 2004; Fig. 5 g). PAE-Tie2-K854R did not support Ang-1dependent increased adhesion, which further suggested that this process was Tie2 dependent.
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Discussion |
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Integrin occupancy may cause growth factor receptor autophosphorylation (Moro et al., 1998), and several data demonstrate that growth factor receptors and integrins associate under growth factor stimulation (Woodard et al., 1998; Soldi et al., 1999; Lee and Juliano, 2002) or under integrin activation (Beningo et al., 2001; Baron et al., 2002). In ECs, Tie2 is present in free-form and associated with 5ß1 integrin. The activation of
5ß1 integrin by fibronectin increases the complex with Tie2 and modulates the time and concentration window of the receptor activation. When
5ß1 is activated, Tie2 is phosphorylated at low Ang-1 concentrations, whereas at higher Ang-1 concentrations the activation is attenuated. Moreover,
5ß1 expression influences the duration of Ang-1dependent Tie2 phosphorylation. Therefore, we may speculate that
5ß1 activation could influence Tie2 signal duration and signal strength. It has been reported how the quantitative modulation of signal threshold of tyrosine kinase receptors affects the biologic outcome (Hunter, 2000).
It has been suggested that cooperation between integrins and growth factor receptors may be due to the formation of a complex between FAK and the cytosolic domain of the receptor tyrosine kinase (Sieg et al., 2000). Alternatively, it may involve the direct interaction of the receptors (Borges et al., 2000). Our data show that Tie2 and 5ß1 association occurs independently from cell adhesion and
5ß1 activation. Fibronectin-mediated
5ß1 activation up-regulates the constitutive basal level of Tie2/
5ß1 interaction without increasing FAK in the complex. Therefore, these evidences suggest that Tie2 and
5ß1 complex independently from the presence of a FAK-mediated intracellular bridge, and that this association should be direct, as reported for the interaction between VEGFR-2 and
vß3 (Borges et al., 2000) and further stabilized by conformational changes occurring during integrin activation.
Peculiarly, Ang-1 does not modify the features of association between Tie2 and fibronectin-engaged 5ß1, but triggers biochemical signals that recruit p85 and FAK to the complex. It is known that p85 binds activated Tie2 (Kontos et al., 1998), whereas FAK is recruited to the cytosolic tail of clusterized integrins at the focal adhesions (Schlaepfer and Mitra, 2004). Thus, Ang-1 stimulation mediates Tie2 and
5ß1 signaling, and allows a cross-talk between these pathways by acting at the level of Tie2/
5ß1 complex. Integrins respond to intracellular cues by modifying the avidity for their ligands. Growth factor receptors modulate integrin function through inside-out signaling to mediate enhanced biologic effects, such as cell motility, survival, and invasion (Byzova et al., 2000; Guilherme et al., 1998; Baron et al., 2002). Here, we demonstrate that Ang-1/Tie2 activates integrins through the PI3-K signaling which suggests that FAK recruitment to Tie2/
5ß1 complex could be dependent on activated Tie2 inside-out signaling. Conversely, the observation that immobilized (Carlson et al., 2001) or soluble Ang-1 binds and activates
5ß1 in the absence of Tie2 demonstrates that Ang-1 promotes
5ß1 outside-in signaling. Therefore, all together the results suggest a model in which the synergism of Tie-2/
5ß1 inside-out/outside-in signaling would allow the stabilization of the complex and the activation of Tie2 at lower Ang-1 concentrations. Furthermore, increased Tie2/
5ß1 interaction on fibronectin promotes selective signaling pathways that modify the chemokinetic response of ECs to Ang-1 with the appearance of a directional motility.
We demonstrated previously that EC chemokinesis mediated by Ang-1 on native ECM is characterized by two peaks of Rac-1 activation that are PI3-K dependent (Cascone et al., 2003b). In this study, we report that Ang-1 induces a prolonged Rac1 activation over a 2-h stimulation in ECs adhered to fibronectin, but not to other ECM molecules. Rac1-GTP mediates lamellipodia extension at the leading edge and participates in growth factor receptors and integrin signaling pathways (Cho and Klemke, 2002; Burridge and Wennerberg, 2004). Furthermore, ß1 integrin overexpression increases Rac activity (Miao et al., 2002), and PI3-K/Rac1 loop exerts a positive role in directed motility (Wang et al., 2002); this suggests that Tie2/5ß1 cross-talk may enhance Ang-1dependent Rac1 up-regulation, and thus, promote persistent motility.
5ß1 integrin normally is expressed in ECs and is up-regulated during angiogenesis, whereas fibronectin is highly synthesized during embryonic and tissue remodeling and in injured tissues (Armstrong and Armstrong, 2000). Genetic ablation of
5 or fibronectin leads to embryonic lethality with major vascular defects (George et al., 1993; Yang et al., 1993) and Abs or peptides that block
5ß1 function inhibit growth factorinduced angiogenesis (Kim et al., 2000b). Knock-out mice for Ang-1 or Tie2 show severe defects in vascular remodeling, branching, and maturation (Sato et al., 1995; Suri et al., 1996). Ang-1 has angiogenic effects in many models of postnatal angiogenesis, such as retinal vascularization (Uemura et al., 2002) and hindlimb and myocardial ischemia (Shyu et al., 1998; Chae et al., 2000). In other experimental models, Ang-1 exclusively acts in enhancing VEGF-mediated angiogenic effects (Asahara et al., 1998; Zhu et al., 2002). The role of Ang-1 in tumor angiogenesis seems to be correlated strictly with the tumor model analyzed (Metheny-Barlow and Li, 2003), and is proangiogenic (Shim et al., 2002; Machein et al., 2004) and antiangiogenic (Hawighorst et al., 2002; Stoeltzing et al., 2003). In this study, we show that Ang-1 induces vessel remodeling in CAM assay, and that this process depends on
5ß1 integrin and the cell-binding domain of fibronectin, but not on
vß3 integrin. Interestingly, in the same in vivo model, antagonists of
5ß1 integrin blocked bFGF-, TNF-
, and IL-8stimulated angiogenesis, but had a minimal effect on VEGF-A response (Kim et al., 2000b). These evidences indicate that Ang-1 elicits angiogenesis through a strict cooperation with activated
5ß1 integrin. Functional cross-talk between Tie2 and
5ß1 is highlighted by similarities in the defects of heart development in mice that were genetically modified for
5 and Ang-1 or Tie2 (Yang et al., 1993; Dumont et al., 1994; Suri et al., 1996). Expression of
5ß1 in ECs correlates with angiogenesis, and is engaged in ECM remodeling sites where fibronectin and other ECM components accumulate to mediate dynamic interactions with activated ECs (Kalluri, 2003). Recently, Thurston et al. (2005) described how Ang-1 promotes the enlargement of vessels in a critical window of vascular plasticity in neonatal mice. Therefore, we point out that Ang-1/Tie2 biologic effects could be regulated by microenvironment changes that characterize the angiogenic switch occurring in tissues. In conclusion, we demonstrate that Tie2 and
5ß1 structurally and functionally cross-talk in vitro and in vivo, and lead to a fine-tuning modulation of the vascular effect of Ang-1.
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Materials and methods |
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Cells were cultured on wells or transwells (Costar) or dishes (Falcon) coated with fibronectin, collagen I, or fibrinogen at the indicated concentrations, and saturated with 3% BSA. Human recombinant Ang-1 and VEGF-A165 were from R&D Systems.
For the antibodies used, see online supplemental materials (available at http://www.jcb.org/cgi/content/full/jcb.200507082/DC1).
Adhesion assay
ECs and PAE cells were starved in 0.5% FCS. 100 cells/µl were plated in the presence of Ang-1 on 96-well microtiter plates, incubated for 30 min at 37°C, washed three times with PBS, fixed in 3.7% glutaraldehyde, and stained with 0.1% crystal violet. In some experiments, JBS5 anti-5ß1 (2 µg/ml), LM609 anti-
vß3 (2 µg/ml), BHA2.1 anti-
2ß1 (5 µg/ml) Abs (Chemicon), or LY294002 (15 µM) were added to cell suspension. This drug concentration did not modify cell viability as assessed by trypan blue exclusion. The absorbance was read at 540 nm in a spectrophotometer for microtiter plate (HT6 7000 Bio Assay Reader, PerkinElmer).
Motility assay
Starved ECs were plated onto 20-mm dishes and allowed to adhere in 0.5% FCS for 1 h at 37°C. Ang-1 or VEGF-A165 was added to the medium, and ECs were observed with an inverted microscope equipped with thermostatic and CO2 controlled chamber (model DM IRB HC; Leica). Phase-contrast video images of control and stimulated ECs were recorded at 5-min intervals for 4 h using a CCD camera (Hamamatsu Photonics), and were analyzed for velocities of cell migration, path length covered, and directionality of cell motility using DIAS image processing software (Solltech) as described previously (Serini et al., 2003b). Speed, net path length, and persistence parameters of 50 cells from five experiments were calculated and plotted.
Immunoprecipitation
Cells were washed with cold PBS and lysed in buffer with added protease and phosphatase inhibitors (50 µg/ml pepstatin, 50 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 100 µM ZnCl2, 1 mM Na3VO4). To immunoprecipitate integrin complexes, a buffer containing 50 mM HEPES (pH 7.4), 5 mM EDTA, 2 mM EGTA, 150 mM NaCl, 10% glycerol, and 1% NP-40 was used. For all of the other conditions of immunoprecipitation, a buffer containing 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100, 10% glycerol was used. In some experiments, cells were labeled for detection of cell surface proteins (ECL Protein Biotinylation Module, Amersham Biosciences), washed three times with ice-cold PBS, and incubated with bicarbonate buffer containing biotinamidocaproato N-hydroxysuccinamide ester for 30 min at 4°C before lysis. Lysates (1 mg) were precleared with nonimmune goat serum or mouse IgG and protein G-Sepharose (Amersham Biosciences) for 2 h at 4°C and then incubated with protein G-Sepharose or protein A-Sepharose and Abs indicated for 2 h at 4°C. In re-immunoprecipitation experiments, the immune complexes from anti-5ß1 immunoprecipitation were resuspended in 50 mM Tris-HCl pH 7.4, 2% SDS and heated for 5 min at 95°C. Supernatants were diluted 10-fold with lysis buffer and immunoprecipitated with anti-Tie2 Ab. After washes, immunoprecipitates were resolved on SDS-PAGE, immunoblotted as indicated or probed with peroxidase-conjugated streptavidin, and detected by the enhanced chemiluminescence technique (PerkinElmer). Densitometry represent the results of at least five independent experiments.
Chick CAM angiogenesis assays
For CAM assay, we produced some changes to the previously described method (Valdembri et al., 2002). Fertilized chick embryos were incubated for 3 d at 37°C at 70% humidity. A small hole was made over the air sac at the end of the egg and a second hole was made directly over the embryonic blood vessels. After 10 d, cortisone acetatetreated filter disks (5 mm) saturated with 50200 ng of Ang-1 or saline were placed on the CAM in an area with a minimum of small blood vessels. The day after, anti-5ß1 (JBS5; 15 µg), anti-
vß3 (LM609; 10 µg), or anti (anti-CBD; 784A2A6; 25 µg) function-blocking Abs or IgG (25 µg) was applied on the filter disks, and eggs were incubated for 2 d. The concentrations were chosen based on previously published experiments (Kim et al., 2000b).
CAM analysis
CAMs were fixed with PBS-3.7% paraformaldehyde for 10 min at room temperature, filter disks were excised, and pictures were taken with a JVC TK-C1380E color video camera (ImageProPlus 4.0 imaging software) connected to the stereomicroscope (model SZX9; Olympus). Pictures were processed with the imaging software winRHIZO Pro (Regent Instruments Inc.). This software reproduces vessel pattern (Fig. 6 g), identifies vessel branching, and gives back the forks (blood vessel branch points) pro area (arrows in Fig. 6 h). Data represent the results of five independent experiments, each performed in triplicate.
Statistical analysis
Statistical analysis was performed using the unpaired t test or one-way analysis of variance and Bonferroni's test for pairwise multiple comparisons (SSPS 13.0; SPSS Inc.).
Online supplemental material
Fig. S1 shows EC survival data. Fig. S2 shows the colocalization of Tie2 and 5ß1. Fig. S3 shows haptotaxis data. Online supplemental material available at http://www.jcb.org/cgi/content/full/jcb.200507082/DC1.
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Acknowledgments |
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F. Bussolino belongs to the European Vascular Genomics Network (http://www.evgn.org), a Network of Excellence supported by the European Community's Sixth Framework Programme for Research Priority 1 "Life sciences, genomics and biotechnology for health" (Contract number LSHM-CT-2003-503254). This work was supported by grants from Regione Piemonte, Associazione italiana per la Ricerca sul Cancro (A.I.R.C), M.I.U.R. (PRIN 2004; F.I.R.B.: RBNE018P4, RBNE01MAWA, RBNE01T8C8), Istituto Superiore di Sanità (Progetto nazionale AIDS) Ministero della Salute Ricerca finalizzata 2003, and Telethon Italy (grant no. GGP04127 to G. Serini). I. Cascone was supported by (A.I.R.C), L. Napione by "Compagnia di San Paolo."
Submitted: 18 July 2005
Accepted: 8 August 2005
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