Real-time quantitative PCR was used to accurately detect the changes of SHP-2 and GAPDH gene copies. The cycle at which the amplification plot crosses the threshold (CT) is known to accurately reflect relative mRNA values. Total RNA (1µg) DNA-free was prepared and reverse transcribed into cDNA using 1 µg of hexamers (Roche Diagnostics, Meylan, Grenoble) and M-MLV reverse transcriptase as described by the manufacturer (Invitrogen Life Technologies). Human SHP-2 and GAPDH mRNAs were amplified (SHP-2: forward primer, 5’-GAACATGACATCGCGGAGA-3’ and reverse primer 5’-AAAACTGCCATCAACTCCTCTT-3’; GAPDH: forward primer, 5’-CAAATTCCATGGCACCGTC-3’ and reverse primer 5’-CCCATCTGATTTTGGAGGGA-3’), detected and quantitated in real-time using the Fast Start universal SYBR Green Master kit (Roche Diagnostics, Meylan, France) and the LC-480 Detector system (Roche Diagnostics) as described previously (7). PCRs were performed in a 50-µl volume, with 20 mmol/L of Tris-HCL (pH 7.4, 25°C), 50 mmol/L of KCL, 1.5 mmol/L of MgCL₂, 200 µmol/L each of four dNTPs, 1 µmol/L of each primer, cDNA derived from the equivalent of 150 ng of total RNA, and 2.5 U of Taq Polymerase (Roche Diagnostics, Meylan, France). Samples were subjected to 35 cycles. Cycle parameters were generally as follows: the initial denaturation step was at 94°C for 4 minutes, the repeat cycle consisted of annealing at 55°C for SHP-2 and 60°C for GAPDH for 40 seconds, followed by extension at 68°C for 50 seconds and denaturation at 94°C for 30 seconds; the last extension time was lengthened to 10 minutes. The reaction produced a 90-bp PCR product for SHP-2, and one of 101-bp for GAPDH. To determine the accuracy of the assay, total RNA was reverse-transcribed and amplified on 3 separate days. The interassay accuracy of amplification for the 3 days was 8%. For quantitation of the data, SHP-2 mRNA levels were normalized to the GAPDH mRNA levels in the same reaction. PCR experiments were carried out in triplicate (n = 4). Results are expressed as n-fold differences in target gene expression relative to the housekeeping gene (GAPDH).

HUVECs and GECs were incubated under different conditions and for various times as indicated in the Results. Cell extracts were prepared by cell lysis in cold RIPA buffer (150 mM NaCl, 1% NP-40, 0.15% Na-deoxycholate, 1 mM EDTA, 0.1% SDS, and 50 mM Tris, pH 8.0) supplemented with a 1X protease inhibitor mix and phosphatase inhibitor cocktail (Complete Mini Protease Inhibitor cocktail tablets, Roche Diagnostics, Meylan, France). After 10 min of incubation on ice, cell lysates were collected and centrifuged at 10,000 rpm for 10 min at 4°C and processed for Western blot analysis as described (13). Samples were then analyzed by 10% SDS-PAGE. Proteins were transferred to a 0.2 µm nitrocellulose membrane (Whatman, Dassel, Germany), which was subsequently blocked with a blocking buffer containing 5% (wt/vol) milk powder in Tris-buffered saline with Tween 20 (TBST). When phosphospecific antibodies were used, blots were blocked with 5% (wt/vol) bovine serum albumin (BSA) in TBST. The nitrocellulose membrane was incubated with specific primary antibodies VE-cadherin (Santa Cruz, # sc-6458, 1:1,000), pVE-cadherin^Y731 (US Biological, # 226637, 1:1,000), SHP-2 (BD Biosciences, # 610621), pSHP-2^Y542 (Cell Signaling Technology, # 3751, 1:1,000), β-actin (Santa Cruz, # sc-47778, 1:1,000) overnight at 4°C, followed by incubation with secondary HRP-labeled antibodies for 1 h at room temperature. Between the incubation steps, blots were washed with TBST. Staining was visualized with enhanced chemiluminescence (ECL) detection system (Invitrogen Life Technologies Inc.). All the antibodies used in this study were characterized through their use by different laboratories and shown to be specific to their peptide targets. Independent membranes were used for probing for each of the antibodies. Images were then quantified using Image J Software.

For immunoprecipitation, treated HUVECs and GECS with different conditions were lysed in ice-cold lysis buffer (50 mM Tris, 150 mM CaCl₂, 10 mM MgCl₂, 1% Triton-X100, 0.1% SDS, 0.25% deoxycholic acid, pH 7.4), supplemented with phosphatase inhibitor cocktail (Roche Diagnostics) and fresh protease-inhibitor-mixture tablets (Roche Diagnostics). After 10 min of incubation on ice, cell lysates were collected and centrifuged at 10,000 rpm for 10 min at 4°C. Protein concentration of the supernatant was then determined, and antibodies against VE-Cadherin (Santa Cruz, # sc-6458, 5 μg) or SHP-2 (BD Biosciences, # 610621, 5 μg) were added to the same amount of supernatant and incubated for 2h at 4°C, then 30 µl of protein G-Sepharose was added for 1 h at 4°C under continuous mixing. Subsequently, beads were centrifuged at 5000 rpm for 20 s at 4°C, washed five times with lysis buffer, and boiled in SDS-sample buffer (60 µl) containing 4% β-mercaptoethanol. Samples were then analyzed by 10% SDS-PAGE. Proteins were transferred to a 0.2 µm nitrocellulose membrane (Amersham, GE Healthcare Life Sciences) which was subsequently blocked with blocking buffer containing 5% (wt/vol) milk powder in Tris-buffered saline with Tween 20 (TBST). When phosphospecific antibodies were used, blots were blocked with 5% (wt/vol) bovine serum albumin (BSA) in TBST. The nitrocellulose membrane was incubated with specific primary antibodies (anti-VE-cadherin, anti-pVE-cadherin^Y731, anti-SHP-2, and anti-pSHP-2Y⁵⁴²) overnight at 4°C, followed by incubation with secondary HRP-labeled antibodies for 1 h at room temperature. Between the incubation steps, blots were washed with TBST. Staining was visualized with an enhanced chemiluminescence detection system (Invitrogen Life Technologies Inc.).

For RNA interference of SHP-2 expression, the following siRNAs were used: (siSHP-2/1) 5’-CGCUCAUGACUAUACGCUAtt-3’ and (siSHP-2/2) 5’-CAAUGACGGCAAGUCUAAAtt-3’ (Ambion). For negative controls, a siRNA was used that does not target any known mammalian gene (5’-UUCUCCGAACGUGUCACGU-3’; QIAGEN). Routinely, 10⁶ HUVECs were transfected with 6 µg siRNA using nucleofection (Amaxa Biosystems) according to the manufacturer’s instructions.

To determine paracellular permeability, 1.25 x 10⁵ HUVECs were seeded per 6.5-mm-diameter Transwell filters (upper compartment) (Costar 3413; corning) with a 0.4 µm pore size, coated with 25 µg/ml fibronectin as previously described (28). After nucleofection with SHP-2 and control siRNAs, HUVECs were cultured for 24 h before the assay. For pharmacological inhibition, HUVECs were pre-incubated for 3 h with 30 µM NSC-87877 or 30 µM SHP099. Thereafter, AM (10^-9 M) was added to the incubation medium (upper compartment), and after pretreatment for 24 h, 0.25 mg/ml FITC-dextran (250 kD; Sigma Aldrich) was added to the upper compartment. Treatment with VEGF-A (50 ng/ml) was used as positive control known to increase the permeability of endothelial cells (29). After incubation for an additional 30 minutes, 100 µl of the medium was collected from the lower compartment and fluorescence was evaluated with a fluorimeter at 570 nm using a Microplate fluorometer (microplate reader; POLARstar Omega, BMG LABTECH). An endothelial cell monolayer permeability index was calculated as described previously (30).

The morphogenesis assay on Matrigel was performed as previously described (31). HUVECs (7 x 10⁴) were pre-incubated with SHP-2 inhibitors NSC-87877 (30 µM) or SHP099 (30 µM) 30 min before being seeded on Matrigel-precoated wells with 300 µl of 8.5 mg/ml of Matrigel solution (BD Biosciences). For this tube-formation assay, a medium containing 0.5% FBS was supplemented with AM (10^-7 M) in the absence or presence of SHP-2 inhibitors. The plates were then incubated for 5 h at 37°C, and subsequently fixed with methanol-free 4% paraformaldehyde and microscopic images were collected for analysis of junction and node formation. The extent of node formation was quantitatively assessed using Angiogenesis Analyzer plug-in for Image J.

HUVECs were cultured under the same conditions as described under angiogenesis assay and the effects on cell viability of AM, SHP-2 inhibitors (NSC-87877 and SHP099) after 24 h incubation, and siSHP-2 after 48 h treatment were assessed by MTT assay (Promega, Lyon, France).

Female C57BL/6 mice were injected subcutaneously above the rectus abdominus with 700 µl of Matrigel solution (8.5 mg/ml) (Becton Dickinson, France), combined with AM at 2 μg/ml (n = 5) or AM in presence of SHP-2 inhibitor, NSC-87877 (15 μM) (n = 5), or alone as a negative control (n = 5). Three weeks later, the animals of each group were sacrificed and the Matrigel plugs were dissected and fixed in methanol-free 4% paraformaldehyde for histological analysis. Paraffin-embedded sections were processed and stained using hematoxylin and eosin (H&E). Investigators “blinded” to sample identity photographed eight central sections. Immunofluorescence analysis was performed on paraffin-embedded sections. Thin (5 μm) sections were incubated with anti-von Willebrand factor (Dako, #A0085, 1:200) and anti-α-SMA (Dako, # M0851, 1:80) antibodies, and subsequently with a fluorochrome (Alexa 488 or Alexa 562)-conjugated secondary antibodies (Invitrogen Life Technologies).

Animal work was carried out in the animal facility of the School of medicine according to the institutional animal welfare guidelines. Athymic NMRI (nu/nu) nude mice (HARLAN, France) were maintained in a sterile environment with a daily 12-hour light/12-hour dark cycle. The subcutaneous tumors were generated by injection of U87 glioblastoma cells (2 x 10⁶) in the right flank of mice. Tumors were measured with a dial-caliper, and volumes were determined using the formula width x length x height x 0.5236 (for ellipsoid form; 11). At a tumor volume of approximately 300-350 mm³ animals were randomly divided into two groups (n = 6). Two independent experiments were performed with 6 animals in each group that received a daily intraperitoneal injection of pre-immune serum of irrelevant specificity (25 mg/kg of purified IgG/mouse), or αAMR antibodies (25 mg/kg of purified IgG/mouse) for 10 days (13). Mice were sacrificed at the indicated time. The adrenomedullin receptors polyclonal antibodies were developed against CLR, RAMP2, and RAMP3 as reported previously (11) and characterized as described ( Supplementary Figure S2 ). On days 5 and 10 the animals were euthanized and tumors excised and homogenized using RIPA buffer to prepare protein extracts for immunoprecipitation and immunoblotting.

For statistical analysis, non-parametric analysis Kruskal-Wallis followed by Bonferroni test was performed using XLSTAT Software throughout the whole manuscript. Results in bar graphs are given as mean values and their corresponding standard deviation (SD). For all tests, differences were considered statistically significant when *, p < 0.05; **, p < 0.01; ***, p < 0.001.

In a previous study, we showed that AM-blockade induces phosphorylation of VE-cadherin^Y731 in HUVECs (13). Hereby, we demonstrated that after 3 h of incubation with AM (10^-9 M), a significant (p < 0.001) decrease of phosphorylation of VE-cadherin^Y731 in HUVECs is observed, which is sustained over 16 h (p < 0.01) ( Figures 1A, B ). In agreement with this data, the AM antagonist AM_22-52 induces a substantial increase (p < 0.001) of pVE-cadherin^Y731 ( Figures 1C, D ). Taken together, these data demonstrate that AM is involved in the control of phosphorylation status of VE-cadherin at its critical Tyr⁷³¹ residue.

The dephosphorylation of pVE-cadherin^Y731 after AM treatment can be attributed to elevated tyrosine phosphatase activity. Taking into account that SHP-2 binds to VE-cadherin/β-catenin (14), we hypothesized that this phosphatase might play an essential role in mediating AM activity. Phosphorylation of SHP-2 at tyrosine 542 (pSHP-2^Y542) was previously shown to induce a conformational change that exposes the catalytic domain involved in signaling functions (32, 33). In line with this, a 1-h treatment with AM resulted in increased levels (p < 0.01) of pSHP-2^Y542 in HUVECs, which were maintained for at least 16 h (p < 0.001) ( Figures 2A, B ). On the other hand, pSHP-2^Y542 levels significantly (p < 0.01) decreased after treatment of HUVECs with AM_22-52 antagonist (10^-6 M) for 1 h ( Figures 2C, D ). These changes in the levels of pSHP-2^Y542 responded to modulation of SHP-2 activity and are not a consequence of alterations in its expression levels after AM or AM_22-52 treatments ( Figures 2C, D ). Furthermore, no significant differences in SHP-2 mRNA levels were observed at a different time point, subsequently to AM- or AM_22-52-treatment when compared to untreated cells ( Figure 2E ).

To evaluate the involvement of pSHP-2^Y542 in AM-mediated dephosphorylation of pVE-cadherin^Y731, we employed phosphatase pharmacological inhibitors (NSC-87877 which targets SHP-1 and SHP-2 and SHP099 which inhibits specifically SHP-2) and small interfering RNA (siRNA). As shown in Figure 3 , dephosphorylation of pVE-cadherin^Y731 by AM in HUVECs was significantly reduced when they were pre-incubated with increasing amounts of NSC-87877 (p < 0.01) ( Figures 3A, B ) or SHP099 (p < 0.01) ( Figures 3C, D ), indicating that pSHP-2^Y542 is responsible for the pVE-cadherin^Y731 dephosphorylation and that AM controls the level of pVE-cadherin^Y731 by activating SHP-2. The data demonstrate that 30 µM of SHP099 overshoot the inhibitory effect against AM and VE-cadherin is more phosphorylated than control, suggesting that SHP099 at 30 µM was efficient to inhibit the SHP-2 activity induced by both the exogenous AM (10^-7 M) and the AM secreted by the HUVECs that act via autocrine/paracrine loop. Furthermore, HUVECs were transfected with two SHP-2 siRNA (siSHP-2/1 and siSHP-2/2) able to reduce SHP-2 levels by up to 80% with respect to control siRNA ( Figure 3E ). In these cells, no dephosphorylation of pVE-cadherin^Y731 ( Figures 3E, F ) can be observed after AM treatment.

Taken together, the findings indicate that SHP-2 phosphatase activity is required for dephosphorylation of pVE-cadherin^Y731 upon stimulation with AM.

Reduction of VE-cadherin from the cell-cell adhesion sites, and tyrosine phosphorylation of VE-cadherin are mechanisms known to be responsible for cell-cell contact disruption (34). We examined whether AM plays a role in the association of SHP-2 with VE-cadherin or in the activation of SHP-2 once it is already associated to VE-cadherin. Accordingly, VE-cadherin was immunoprecipitated from lysates of control cells, AM_22-52-treated endothelial cells, and AM-treated endothelial cells in the presence or absence of SHP-2 inhibitors. Subsequently, the immunoprecipitants were processed for immunoblotting using anti-SHP-2, anti-pSHP-2^Y542, anti-pVE-cadherin^Y731 and anti-VE-cadherin antibodies ( Figure 4A ). Nearly equal amounts of SHP-2 were immunoprecipitated from control and treated HUVECs, suggesting that SHP-2 associates to VE-cadherin independently of AM signaling ( Figures 4A, B ). Western blot analysis using anti-pSHP-2^Y542 antibody showed a significant increase (p < 0.05) of pSHP-2^Y542 associated to VE-cadherin in AM-treated cells ( Figures 4A, C ) when compared to control cells, while upon treatment with AM_22-52, pSHP-2^Y542 levels associated to VE-cadherin were not modified ( Figures 4A, C ). Similar results were obtained after pre-treatment with SHP-2 inhibitors ( Figures 4A, C ), demonstrating that both compounds (NSC-87877 and SHP099) block the AM-induced phosphorylation for SHP-2^Y542 associated with VE-cadherin. Immunoblotting with anti-pVE-cadherin^Y731 antibody demonstrated a significant decrease (p < 0.05) of pVE-cadherin^Y731 in HUVECs treated with AM ( Figures 4A, D ), which was lost upon incubation of HUVECs with SHP-2 inhibitors or AM_22-52 ( Figures 4A, D ). Collectively, these data demonstrate that SHP-2 not only forms an enzyme-substrate complex with VE-cadherin independently of AM but also regulates VE-cadherin dephosphorylation upon stimulation by AM.

The relevance of SHP-2 in the maintenance of a physiological endothelium upon treatment with AM was evaluated by the passage of 250 kD FITC-dextran through HUVECs monolayer treated with siSHP-2/1, siSHP-2/2, NSC-87877 or SHP099. As shown in Figures 5A, B , AM significantly decreased (p < 0.01) the cell layer permeability by 30% to 40% as compared with the control. Incubation of the cells with NSC-87877, SHP099, or siSHP-2 siRNAs blocked the AM-induced decrease of the cell layer permeability with a significant increase (p < 0.05) of permeability observed under NSC-87877 (inhibits both SHP-1 and SHP-2), which exceeds the control levels, that could be due to the inhibition of SHP1 pointing out the putative implication of SHP1 in the permeability assay in the control cells. By incubating cells with AM and NSC-87877, the increase of permeability is lost and reached the levels observed in control cells suggesting that AM may act to interfere somehow with the pathway that involves SHP1. Of note, the effect of siSHP-2/2 appears stronger than the one obtained by the siSHP-2/1. The permeability in presence of siSHP-2/2 is significantly increased (p < 0.05) as compared to control cells suggesting that the effect of endogenous AM by autocrine and/or paracrine loop on permeability is inhibited upon inactivation of SHP-2 ( Figure 5B ). This indicates that the SHP-2 activity is required for the maintenance of cell contact integrity in HUVECs upon stimulation with AM. Treatment with VEGF-A demonstrated a significant increase (p < 0.05) of HUVECs permeability ( Figure 5A ) as previously reported (29).

To investigate whether the effects seen after SHP-2 inhibition have functional consequences on AM-induced vessel formation, we firstly performed Matrigel assays. In a previous work, we showed the ability of AM to induce HUVECs to form capillary-like vascular structures in an in vitro Matrigel assay (31). In the presence of 0.5% FBS, partial capillary-like structures formed by HUVECs can be observed ( Figure 5C ). After addition of AM (10^-9 M), HUVECs developed a more robust capillary-like structures (p < 0.01) compared to control cells ( Figure 5C ). Incubation of HUVECs with inhibitors NSC-87877 or SHP099 impairs the AM-induced tube formation (p < 0.05; p < 0.01, respectively) and induced a drastic reduction in the number of endothelium-dependent capillary-like structures ( Figure 5C ), as indicated by the quantification of the capillary-like structures ( Figure 5D ). The capillary-like structures were incomplete compared to AM-treated cells ( Figure 5C ). In Figures 5A, C , the effect of SHP099 is differ between permeability (A) and tube formation (C). The difference in the effect might be due to the inhibition of SHP-2 involved in pathways activated and/or inhibited subsequently to the interactions of the HUVECs with environment, which is different in the context of permeability assay compared to the one of the Matrigel for the angiogenesis assay in vitro. In Figures 5C, D , SHP099 is more potent than NSC-87877 disrupting AM-induced tube formation as compared to control cells and treated cells with AM or AM + NSC-87877. These results suggest that activation of SHP-2 is critical for the AM proangiogenic activity with a major role in the AM-induced tube formation in vitro. To rule out the possibility of the AM, NSC-87877, SHP099, and SHP-2 siRNAs being cytotoxic and therefore to contribute to the observed effects, we performed a cell viability assay with cells treated with AM, SHP-2 inhibitors and SHP-2 siRNAs. No significant cell death can be observed with the reported experimental conditions ( Supplementary Figures S3A, B ). This confirms that the effects seen after AM treatment or SHP-2 inhibition are indeed specific and not due to cytotoxicity from the solvent.

We previously demonstrated a high expression of AM in GBM and its role to maintain a stable and functional vascularization in this disease (7, 11, 35). Thus, based on this and aiming to corroborate in this valuable cell model results shown above, the AM capacity to modulate VE-cadherin phosphorylation status through pSHP-2^Y542 was assayed in GECs. As it is shown in Figures 6 , after 6 h of exposure of the cells to AM (10^-9 M), pVE-cadherin^Y731 decreased significantly (p < 0.05) ( Figures 6A, B ) over time concomitantly with an increase in pSHP-2^Y542 (p < 0.05; p < 0.01) ( Figures 6E, F ). As expected, AM_22-52 treatment substantially (p < 0.01) elevated the levels of pVE-cadherin^Y731 ( Figures 6C, D ) while decreased (p < 0.01) pSHP-2^Y542 ( Figures 6G, H ).

Similar results were obtained for the second set of GBM-associated ECs ( Supplementary Figure S4 ). Furthermore, no significant differences in SHP-2 mRNA levels could be observed at different time points subsequently to AM- or AM_22-52-treatment when compared to untreated cells ( Supplementary Figure S5 ).

We also demonstrated that the VE-cadherin immunoprecipitates from GECs showed equal amounts of total SHP-2 associated with VE-cadherin independently of AM-treatment ( Figures 6I, J ). A significant increase (p < 0.05) in pSHP-2^Y542 associated with VE-cadherin was observed in AM incubated cells while pSHP-2^Y542 showed to be substantially decreased in AM_22-52 -treatments ( Figures 6I, K ). In agreement with our previous results, AM induced a marked diminution (p < 0.001) in pVE-cadherin^Y731 levels accompanied by a significant increase (p < 0.05) in pVE-cadherin^Y731 after AM_22-52 treatments ( Figures 6I, L ).

The magnitude of the effect that SHP-2 inhibition may have in the AM-induced angiogenesis was evaluated in vivo by employing the Matrigel plug assay. Histochemical analysis of control Matrigel plugs exhibited <2% of cellularity ( Figure 7A, a ), while Matrigel plugs in presence of AM revealed a significant increase (p < 0.001) of cell recruitment ( Figures 7A, b ). This was remarkably reduced (p < 0.001) by NSC-87877 ( Figures 7A, c ). vWF staining of AM-plugs sections showed the presence of strong vascular channels lined by endothelial cells ( Figures 7B, b ) with important vessel areas ( Figures 7B, b , inset). α-SMA staining demonstrates that these structures are reinforced and stabilized by the recruitment of vascular smooth muscle cells ( Figures 7B, b ). The reduction in the AM-induced angiogenic phenotype micro-vessel density by > 96% in presence of NSC-87877 indicates the involvement of pSHP-2^Y542 in mediating this process ( Figures 7B, c ). These results demonstrated that AM promotes the recruitment of endothelial-like cells and smooth muscle-like cells, hence; maintaining transient micro-vessel stability ( Figures 7B, b ). This was corroborated by a significant decrease (p < 0.001) in vWF and α-SMA stained cells in the Matrigel ( Figures 7B, c ). The mean vessel area was significantly higher (p < 0.001; p < 0.01) in the Matrigel with AM when compared to Matrigel with AM combined with SHP-2 inhibitor and control Matrigel ( Figure 7C ). In all, these results clearly evidence that SHP-2 activity is required for the AM-transduced signaling to build up stable neovascularization.

To determine whether AM strengthens endothelial cells adhesions via pSHP-2^Y542 in neoplastic nascent vessels; animals bearing U87 tumors were daily treated with intraperitoneal injection with control IgG (600 μg; control group) or αAMR antibodies (600 μg) for 10 days. At this point, the αAMR treatment showed a substantial reduction (p < 0.01) in tumor masses with respect to control ( Figure 8A ). This effect was associated with less vascularization with clear depletion of endothelial cells, as indicated by immunostaining with anti-vWF antibody ( Figures 8B, b ). In contrast, control IgG-treated U87 tumors showed a well-organized vascularization ( Figures 8B, a ). To evaluate the SHP-2/VE-cadherin association, SHP-2 and VE-cadherin were immunoprecipitated from protein extracts prepared from control IgG- and αAMR-treated tumors at days 5 and 10. Both immunoprecipitants demonstrated a reciprocal association between SHP-2 and VE-cadherin, which was not disrupted by αAMR treatment ( Figures 8C, D, G ). This suggests that AM is not involved in the regulation of VE-cadherin/SHP-2 association in vivo. In addition, a significant decrease (p < 0.05; p < 0.01) of pSHP-2^Y542 levels was detected in αAMR-treated tumors compared to control IgG-treated tumors ( Figures 8C, E, H ). Furthermore, immunoblotting with anti-pVE-caderin^Y731 antibody showed a significant increase (p < 0.05; p < 0.01) of pVE-cadherin^Y731 in αAMR-treated tumors with respect to control IgG-administered tumors ( Figures 8C, F, I ).

Taken together, these results proved the involvement of AM to maintain SHP-2 under its active form (pSHP-2^Y542) to dephosphorylate pVE-cadherin^Y731 in vivo to maintain endothelial cell barrier integrity thereby promoting a stable and functional tumor vasculature.