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.
Highlights
Stability and functional assembly of tumor neovessels are governed by collaboration of multiple organ-specific cellular and angiogenic factors
To gain more insight into the mechanism(s) causing neovessels destabilization subsequently to αAM, αAMR, and AM22–52 treatment, we hypothesized that AM blockade might interfere through the endothelial cell junctions somehow to destabilize the tumor neovessels
The immunohistochemical analysis of αAM and αAMR-treated tumors showed a clear decrease in microvessel density with 80% reduction of endothelial cells (Figure 1B)
Summary
Stability and functional assembly of tumor neovessels are governed by collaboration of multiple organ-specific cellular and angiogenic factors. Targeted genetic manipulation and antibody-mediated inhibition of angiogenic growth factors and their receptors in murine tumor models have resulted in identification of key angiogenic modulators that support tumor neoangiogenesis. AM binds to and mediates its activity through the G protein-coupled receptor calcitonin receptor-like receptor (CLR), with specificity for AM being conferred by the receptor activity modifying protein -2 (RAMP2) and -3 (RAMP3) [2]. The use of targeted mouse models clearly indicates that functional AM signalling is essential for embryonic survival. The genetic ablation of Adm [4, 5], calcrl [6], Ramp2 [7,8,9] or the enzyme responsible for functional AM amidation, peptidylglycine alpha-amidating monooxygenase (PAM) [10] all result in midgestational lethality associated with severe interstitial edema and cardiovascular defects
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