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Adrenomedullin blockade induces regression of tumor neovessels through interference with vascular endothelial-cadherin signalling.

Khalfaoui-Bendriss G, Dussault N, Fernandez-Sauze S, Berenguer-Daizé C, Sigaud R, Delfino C, Cayol M, Metellus P, Chinot O, Mabrouk K, Martin PM, Ouafik L - Oncotarget (2015)

Bottom Line: At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function.Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin.In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, CRO2, UMR_S 911, Faculté de Médecine, Marseille, France.

ABSTRACT
The cellular and molecular mechanisms by which adrenomedullin (AM) blockade suppresses tumor neovessels are not well defined. Herein, we show that AM blockade using anti-AM and anti-AM receptors antibodies targets vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and induces regression of unstable nascent tumor neovessels. The underlying mechanism involved, and shown in vitro and in vivo in mice, is the disruption of the molecular engagement of the endothelial cell-specific junctional molecules vascular endothelial-cadherin (VE-cadherin)/β-catenin complex. AM blockade increases endothelial cell permeability by inhibiting cell-cell contacts predominantly through disruption of VE-cadherin/β-catenin/Akt signalling pathway, thereby leading to vascular collapse and regression of tumor neovessels. At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function. Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin. In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation. We found that AM blockade induces β-catenin phosphorylation on Ser33/Ser37/Thr41 sites in both ECs and VSMCs both in vitro and in vivo in mice. These data suggest that AM blockade selectively induces regression of unstable tumor neovessels, through disruption of VE-cadherin signalling. Targeting AM system may present a novel therapeutic target to selectively disrupt assembly and induce regression of nascent tumor neovessels, without affecting normal stabilized vasculature.

No MeSH data available.


Related in: MedlinePlus

αAM and αAMR increase endothelial cell permeability in vitroModification of endothelial cell permeability of αAM and αAMR-treated HUVEC monolayers was assessed as described in Materials and Methods at the indicated time points. Results of three independent experiments in triplicate are presented. Values are means ± SEM. Where indicated, statistical analysis was performed with 1-way ANOVA followed by PLSD test, and the level of significance was set at P < 0.05.
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Figure 4: αAM and αAMR increase endothelial cell permeability in vitroModification of endothelial cell permeability of αAM and αAMR-treated HUVEC monolayers was assessed as described in Materials and Methods at the indicated time points. Results of three independent experiments in triplicate are presented. Values are means ± SEM. Where indicated, statistical analysis was performed with 1-way ANOVA followed by PLSD test, and the level of significance was set at P < 0.05.

Mentions: To determine whether αAM and αAMR induced loss of cell-cell interaction and disruption of VE-cadherin/β-catenin complex that would result in reduced monolayer integrity, we assessed the effect of αAM and αAMR on the integrity of intercellular junctions by measuring the permeability of a confluent HUVEC monolayer. Confluent monolayers of human endothelial cells cultivated on transwell filter inserts were treated with AM (10−7 M), αAM (70 μg/ml), αAMR (70 μg/ml), and IgG control (70 μg/ml). The permeability of the monolayer for Trypan Blue-BSA was determined at several time points by measuring the absorbance intensity of the medium in the lower compartment (Figure 4). In the absence of addition of AM (Ctrl), or presence of AM or IgG control, the level of Trypan Blue-BSA in the lower compartment was low and did not change during the time of experiments 1, 6 and 24 h (Figure 4). When the cells were treated one time with αAM or αAMR, we observed a significant increase in Trypan Blue-BSA permeability compared to control cells, which was highly significant after a delay of about 6 h and started to decrease by 24 h to reach the control values by 30 h of incubation (Figure 4). These results demonstrate that αAM and αAMR impair the endothelial cell barrier function through dysregulation of VE-cadherin homotypic interaction, leading to increased endothelial cell permeability.


Adrenomedullin blockade induces regression of tumor neovessels through interference with vascular endothelial-cadherin signalling.

Khalfaoui-Bendriss G, Dussault N, Fernandez-Sauze S, Berenguer-Daizé C, Sigaud R, Delfino C, Cayol M, Metellus P, Chinot O, Mabrouk K, Martin PM, Ouafik L - Oncotarget (2015)

αAM and αAMR increase endothelial cell permeability in vitroModification of endothelial cell permeability of αAM and αAMR-treated HUVEC monolayers was assessed as described in Materials and Methods at the indicated time points. Results of three independent experiments in triplicate are presented. Values are means ± SEM. Where indicated, statistical analysis was performed with 1-way ANOVA followed by PLSD test, and the level of significance was set at P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4480698&req=5

Figure 4: αAM and αAMR increase endothelial cell permeability in vitroModification of endothelial cell permeability of αAM and αAMR-treated HUVEC monolayers was assessed as described in Materials and Methods at the indicated time points. Results of three independent experiments in triplicate are presented. Values are means ± SEM. Where indicated, statistical analysis was performed with 1-way ANOVA followed by PLSD test, and the level of significance was set at P < 0.05.
Mentions: To determine whether αAM and αAMR induced loss of cell-cell interaction and disruption of VE-cadherin/β-catenin complex that would result in reduced monolayer integrity, we assessed the effect of αAM and αAMR on the integrity of intercellular junctions by measuring the permeability of a confluent HUVEC monolayer. Confluent monolayers of human endothelial cells cultivated on transwell filter inserts were treated with AM (10−7 M), αAM (70 μg/ml), αAMR (70 μg/ml), and IgG control (70 μg/ml). The permeability of the monolayer for Trypan Blue-BSA was determined at several time points by measuring the absorbance intensity of the medium in the lower compartment (Figure 4). In the absence of addition of AM (Ctrl), or presence of AM or IgG control, the level of Trypan Blue-BSA in the lower compartment was low and did not change during the time of experiments 1, 6 and 24 h (Figure 4). When the cells were treated one time with αAM or αAMR, we observed a significant increase in Trypan Blue-BSA permeability compared to control cells, which was highly significant after a delay of about 6 h and started to decrease by 24 h to reach the control values by 30 h of incubation (Figure 4). These results demonstrate that αAM and αAMR impair the endothelial cell barrier function through dysregulation of VE-cadherin homotypic interaction, leading to increased endothelial cell permeability.

Bottom Line: At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function.Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin.In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation.

View Article: PubMed Central - PubMed

Affiliation: Aix Marseille Université, CRO2, UMR_S 911, Faculté de Médecine, Marseille, France.

ABSTRACT
The cellular and molecular mechanisms by which adrenomedullin (AM) blockade suppresses tumor neovessels are not well defined. Herein, we show that AM blockade using anti-AM and anti-AM receptors antibodies targets vascular endothelial cells (ECs) and vascular smooth muscle cells (VSMCs), and induces regression of unstable nascent tumor neovessels. The underlying mechanism involved, and shown in vitro and in vivo in mice, is the disruption of the molecular engagement of the endothelial cell-specific junctional molecules vascular endothelial-cadherin (VE-cadherin)/β-catenin complex. AM blockade increases endothelial cell permeability by inhibiting cell-cell contacts predominantly through disruption of VE-cadherin/β-catenin/Akt signalling pathway, thereby leading to vascular collapse and regression of tumor neovessels. At a molecular level, we show that AM blockade induces tyrosine phosphorylation of VE-cadherin at a critical tyrosine, Tyr731, which is sufficient to prevent the binding of β-catenin to the cytoplasmic tail of VE-cadherin leading to the inhibition of cell barrier function. Furthermore, we demonstrate activation of Src kinase by phosphorylation on Tyr416, supporting a role of Src to phosphorylate Tyr731-VE-cadherin. In this model, Src inhibition impairs αAM and αAMR-induced Tyr731-VE-cadherin phosphorylation in a dose-dependent manner, indicating that Tyr731-VE-cadherin phosphorylation state is dependent on Src activation. We found that AM blockade induces β-catenin phosphorylation on Ser33/Ser37/Thr41 sites in both ECs and VSMCs both in vitro and in vivo in mice. These data suggest that AM blockade selectively induces regression of unstable tumor neovessels, through disruption of VE-cadherin signalling. Targeting AM system may present a novel therapeutic target to selectively disrupt assembly and induce regression of nascent tumor neovessels, without affecting normal stabilized vasculature.

No MeSH data available.


Related in: MedlinePlus