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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 disengage VE-cadherin and disrupt β-catenin distribution in HUVECs in vitro(A) adherens junctions were assessed by immunofluorescence staining of clustered VE-cadherin molecules. The localization of VE-cadherin in confluent HUVECs monolayers was monitored during 24 h by microscopy. While VE-cadherin molecules are clustered in adherens junction of control and AM (10−7 M)-treated cells, addition of αAM (70 μg/ml) leads to redistribution of VE-cadherin disrupting cell-cell contacts that induce the apparition of holes between the cells (indicated by asterisks). DAPI-stained nuclei are in blue. (B) αAM disrupts the β-catenin distribution. The experiments and analyses for β-catenin were processed as described for VE-cadherin in A. DAPI-stained nuclei are in blue. (C) αAM treatment for 10 h caused a disengagement between cells, a reorganisation of actin fibers stained with phalloidin (green) that become localized around the cell body and β-catenin staining observed in the cytoplasm when compared to IgG-control treated cells. DAPI-stained nuclei are in blue.
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Figure 3: αAM and αAMR disengage VE-cadherin and disrupt β-catenin distribution in HUVECs in vitro(A) adherens junctions were assessed by immunofluorescence staining of clustered VE-cadherin molecules. The localization of VE-cadherin in confluent HUVECs monolayers was monitored during 24 h by microscopy. While VE-cadherin molecules are clustered in adherens junction of control and AM (10−7 M)-treated cells, addition of αAM (70 μg/ml) leads to redistribution of VE-cadherin disrupting cell-cell contacts that induce the apparition of holes between the cells (indicated by asterisks). DAPI-stained nuclei are in blue. (B) αAM disrupts the β-catenin distribution. The experiments and analyses for β-catenin were processed as described for VE-cadherin in A. DAPI-stained nuclei are in blue. (C) αAM treatment for 10 h caused a disengagement between cells, a reorganisation of actin fibers stained with phalloidin (green) that become localized around the cell body and β-catenin staining observed in the cytoplasm when compared to IgG-control treated cells. DAPI-stained nuclei are in blue.

Mentions: Accordingly, HUVECs were incubated with AM (10−7 M), αAM (70 μg/ml), or IgG control (70 μg/ml). During the first hour of treatment, VE-cadherin was focally localized to the interjunctional region of endothelial cells (Figure 3A). After 6 h of treatment with αAM, the VE-cadherin staining at cell-cell contacts became very thin, and redistributed into a disorganized pattern (Figure 3A, arrow), which has been previously shown to be the hallmark of VE-cadherin disengagement [30]. However, at 24 h treatment, the redistributed VE-cadherin at the cell-cell contacts appeared to be associated with the partitioned and separated gaps between the endothelial cells suggesting a loss of intercellular contacts (Figure 3A, asterisks). The rapid initial VE-cadherin redistribution that occurred before cell retraction was concomitant with the redistribution of β-catenin, a signalling partner of VE-cadherin. In fact, staining for β-catenin showed a similar pattern at the same time points leading to the formation of separated gaps (Figure 3B, asterisks). Treatment with αAM induces a clear separation between endothelial cells accompanied with redistribution of actin fibres on the periphery and β-catenin staining localized at the cytoplasm (Figure 3C). The treatment with αAMR and AM22–52 demonstrated the same patterns as αAM (not shown). Incubation of human microvascular endothelial cells (HMEC) with αAM and αAMR in vitro induced cell-cell disruption between endothelial cells as demonstrated for HUVECs (Supplementary Figure S1). Therefore, we focused our attention on the mechanisms by which αAM and αAMR may exert their potent anti-vascular effects through destabilization of the VE-cadherin/β-catenin complex.


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 disengage VE-cadherin and disrupt β-catenin distribution in HUVECs in vitro(A) adherens junctions were assessed by immunofluorescence staining of clustered VE-cadherin molecules. The localization of VE-cadherin in confluent HUVECs monolayers was monitored during 24 h by microscopy. While VE-cadherin molecules are clustered in adherens junction of control and AM (10−7 M)-treated cells, addition of αAM (70 μg/ml) leads to redistribution of VE-cadherin disrupting cell-cell contacts that induce the apparition of holes between the cells (indicated by asterisks). DAPI-stained nuclei are in blue. (B) αAM disrupts the β-catenin distribution. The experiments and analyses for β-catenin were processed as described for VE-cadherin in A. DAPI-stained nuclei are in blue. (C) αAM treatment for 10 h caused a disengagement between cells, a reorganisation of actin fibers stained with phalloidin (green) that become localized around the cell body and β-catenin staining observed in the cytoplasm when compared to IgG-control treated cells. DAPI-stained nuclei are in blue.
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Related In: Results  -  Collection

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

Figure 3: αAM and αAMR disengage VE-cadherin and disrupt β-catenin distribution in HUVECs in vitro(A) adherens junctions were assessed by immunofluorescence staining of clustered VE-cadherin molecules. The localization of VE-cadherin in confluent HUVECs monolayers was monitored during 24 h by microscopy. While VE-cadherin molecules are clustered in adherens junction of control and AM (10−7 M)-treated cells, addition of αAM (70 μg/ml) leads to redistribution of VE-cadherin disrupting cell-cell contacts that induce the apparition of holes between the cells (indicated by asterisks). DAPI-stained nuclei are in blue. (B) αAM disrupts the β-catenin distribution. The experiments and analyses for β-catenin were processed as described for VE-cadherin in A. DAPI-stained nuclei are in blue. (C) αAM treatment for 10 h caused a disengagement between cells, a reorganisation of actin fibers stained with phalloidin (green) that become localized around the cell body and β-catenin staining observed in the cytoplasm when compared to IgG-control treated cells. DAPI-stained nuclei are in blue.
Mentions: Accordingly, HUVECs were incubated with AM (10−7 M), αAM (70 μg/ml), or IgG control (70 μg/ml). During the first hour of treatment, VE-cadherin was focally localized to the interjunctional region of endothelial cells (Figure 3A). After 6 h of treatment with αAM, the VE-cadherin staining at cell-cell contacts became very thin, and redistributed into a disorganized pattern (Figure 3A, arrow), which has been previously shown to be the hallmark of VE-cadherin disengagement [30]. However, at 24 h treatment, the redistributed VE-cadherin at the cell-cell contacts appeared to be associated with the partitioned and separated gaps between the endothelial cells suggesting a loss of intercellular contacts (Figure 3A, asterisks). The rapid initial VE-cadherin redistribution that occurred before cell retraction was concomitant with the redistribution of β-catenin, a signalling partner of VE-cadherin. In fact, staining for β-catenin showed a similar pattern at the same time points leading to the formation of separated gaps (Figure 3B, asterisks). Treatment with αAM induces a clear separation between endothelial cells accompanied with redistribution of actin fibres on the periphery and β-catenin staining localized at the cytoplasm (Figure 3C). The treatment with αAMR and AM22–52 demonstrated the same patterns as αAM (not shown). Incubation of human microvascular endothelial cells (HMEC) with αAM and αAMR in vitro induced cell-cell disruption between endothelial cells as demonstrated for HUVECs (Supplementary Figure S1). Therefore, we focused our attention on the mechanisms by which αAM and αAMR may exert their potent anti-vascular effects through destabilization of the VE-cadherin/β-catenin complex.

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