Unraveling the mechanisms involved in endothelial barrier protective effects of angiopoietin-1 variant MAT.Ang-1

With great interest we read the recent article by Alfi eri and colleagues [1], demonstrating that angiopoietin (Ang)-1 variant MAT.Ang-1 improved endotoxemiainduced microvascular dysfunction and microvascular hyperpermeability. Th e authors suggested that MAT. Ang-1-induced recovery of microcirculatory tissue perfusion during sepsis is due to preservation of endothelial barrier integrity. To further elucidate the mechanism, they investigated the possibility of involve ment of VE-cadherin, a major adherens junctions protein responsible for microvascular leakage in infl am mation. Th ey found, however, while there was no change in overall expression of VE-cadherin, MAT.Ang-1 increased VEcadherin phosphorylation in the treated mice, which appears unable to explain the observed endothelial barrier protective eff ects of MAT.Ang-1. Th e work by Dejana and co-workers [2] highlights the critical role of VE-cadherin for maintenance of endothelial barrier function. It is generally accepted that the tyrosine phosphorylation of VE-cadherin and other components of adherens junctions induced by permeabilityincreasing agents is associated with weak junctions and impaired barrier function via regulating VE-cadherin member localization [2]. Recently, among the nine tyrosines in the cytoplasmic tail of VE-cadherin, Potter and colleagues [3] revealed that tyrosine phosphorylation of VE-cadherin at two critical tyrosines, Tyr-658 and Tyr-731, was suffi cient to disrupt VE-cadherin-mediated cell-cell junctions, leading to inhibition of cell barrier function. Previous studies have shown that Ang-1 restores the endothelial barrier function via phosphorylation-dependent redistribution of VE-cadherin [4,5]. While in the present study the total amount of VE-cadherin was not changed, intriguingly MAT.Ang-1 increases VE-cadherin phosphorylation (at Y658) in sepsis. Th is is unexpected because the endothelial barrier protective eff ects of MAT. Ang-1 do not seem to be consistent with its eff ect on an important cellular junction molecule involved in endothelial cell integrity, namely VE-cadherin; however, other mecha nisms of action cannot be ruled out. Nevertheless, further studies are needed to investigate the mechanisms by which this novel Ang-1 variant rescues the endothelial barrier function.

With great interest we read the recent article by Alfi eri and colleagues [1], demonstrating that angiopoietin (Ang)-1 variant MAT.Ang-1 improved endotoxemiainduced microvascular dysfunction and microvascular hyperpermeability. Th e authors suggested that MAT. Ang-1-induced recovery of microcirculatory tissue perfusion during sepsis is due to preservation of endothelial barrier integrity. To further elucidate the mechanism, they investigated the possibility of involve ment of VE-cadherin, a major adherens junctions protein responsible for microvascular leakage in infl am mation. Th ey found, however, while there was no change in overall expression of VE-cadherin, MAT.Ang-1 increased VEcadherin phosphorylation in the treated mice, which appears unable to explain the observed endothelial barrier protective eff ects of MAT.Ang-1.
Th e work by Dejana and co-workers [2] highlights the critical role of VE-cadherin for maintenance of endothelial barrier function. It is generally accepted that the tyrosine phosphorylation of VE-cadherin and other components of adherens junctions induced by permeabilityincreasing agents is associated with weak junctions and impaired barrier function via regulating VE-cadherin member localization [2]. Recently, among the nine tyrosines in the cytoplasmic tail of VE-cadherin, Potter and colleagues [3] revealed that tyrosine phosphorylation of VE-cadherin at two critical tyrosines, Tyr-658 and Tyr-731, was suffi cient to disrupt VE-cadherin-mediated cell-cell junctions, leading to inhibition of cell barrier function.
Previous studies have shown that Ang-1 restores the endothelial barrier function via phosphorylation-dependent redistribution of VE-cadherin [4,5]. While in the present study the total amount of VE-cadherin was not changed, intriguingly MAT.Ang-1 increases VE-cadherin phosphorylation (at Y658) in sepsis. Th is is unexpected because the endothelial barrier protective eff ects of MAT. Ang-1 do not seem to be consistent with its eff ect on an important cellular junction molecule involved in endothelial cell integrity, namely VE-cadherin; however, other mecha nisms of action cannot be ruled out. Nevertheless, further studies are needed to investigate the mechanisms by which this novel Ang-1 variant rescues the endothelial barrier function.

Alessio Alfi eri, Nicola J Brown and Zoe L Brookes
We appreciate the interest and insightful comments made by Zhang and colleagues concerning our recent research article. Th e functional in vivo studies presented in our manuscript demonstrated that MAT.Ang-1 reduced macromolecular leak and improved tissue perfusion without signifi cantly changing the diameter of microvessels, thus suggesting that the protective eff ects induced by MAT.Ang-1 depend on preserving the endothelial barrier integrity. In addition to the well-recognized role in controlling vascular permeability, VE-cadherin and associated junctional proteins form part of complex signaling cascades regulating important cellular functions [6]. In particular, as discussed in our manu script, dis assembly of the VE-cadherin complex triggers an intra cellular negative signal reducing transendothelial leuko cyte migration in mice 6 hours after challenge with lipo polysaccharide [7]. Th erefore, an increase in VE-cadherin phosphorylation paralleled by reduced inter leukin-1β protein expression may be a mechanism by which MAT.Ang-1 induces protection against micro vascular stasis in sepsis. Furthermore, lipopolysac charide-induced endotoxemia increases the expression of several infl ammatory cytokines (for example, tumor necrosis factor-α), which in turn cause macromolecular leak [8]. Th erefore, in vivo a complex mechanistic scenario develops in sepsis with regards to the endothelial barrier function, which is diffi cult to unravel. Th e elegant studies referenced by Zhang and colleagues concerning Ang-1 and VEcadherin phosphorylation report in vitro fi ndings, whereas all our results are from septic mice in vivo with or without MAT.Ang-1 post-treatment. Never theless, we agree that further investigations are required before making fi rm conclusions on the eff ects of MAT.Ang-1 on the endothelium in sepsis -for instance, studies aimed at providing a complete in vivo time-course of the expression, localization and phosphorylation of endo thelial junctional proteins would be extremely informative.

Competing interests
The authors declare that they have no competing interests.