Endothelial destabilization by angiopoietin-2 via integrin β1 activation.

Laura Hakanpaa,Tuomas Sipila,Guillaume Jacquemet,Johanna Ivaska,Veli-Matti Leppanen,Prson Gautam,Lauri Eklund,Harri Nurmi,Pipsa Saharinen,Kari Alitalo

doi:10.1038/ncomms6962

Abstract

Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases. Angiopoietin-1 (Ang1) supports endothelial stabilization via Tie2 activation. Angiopoietin-2 (Ang2) functions as a context-dependent Tie2 agonist/antagonist promoting pathological angiogenesis, vascular permeability and inflammation. Elucidating Ang2-dependent mechanisms of vascular destablization is critical for rational design of angiopoietin antagonists that have demonstrated therapeutic efficacy in cancer trials. Here, we report that Ang2, but not Ang1, activates β1-integrin, leading to endothelial destablization. Autocrine Ang2 signalling upon Tie2 silencing, or in Ang2 transgenic mice, promotes β1-integrin-positive elongated matrix adhesions and actin stress fibres, regulating vascular endothelial-cadherin-containing cell–cell junctions. The Tie2-silenced monolayer integrity is rescued by β1-integrin, phosphoinositide-3 kinase or Rho kinase inhibition, and by re-expression of a membrane-bound Tie2 ectodomain. Furthermore, Tie2 silencing increases, whereas Ang2 blocking inhibits transendothelial tumour cell migration in vitro. These results establish Ang2-mediated β1-integrin activation as a promoter of endothelial destablization, explaining the controversial vascular functions of Ang1 and Ang2.

Highlights

Angiopoietins regulate vascular homeostasis via the endothelial Tie receptor tyrosine kinases
These results suggest that Ang[2], via b1-integrin activation, may predispose vessels to endothelial destablization
In line with the known role of actin stress fibres in generating a centripetal tension that weakens endothelial cell–cell junctions[30], the adherens junction proteins VE-cadherin and b-catenin were reduced in the cell–cell contacts of Tie2-silenced blood microvascular endothelial cells (BECs) and human pulmonary microvascular endothelial cells (Fig. 1a,d; Supplementary Fig. 2a,b)

Summary

Results and Discussion

Tie[2] silencing reduces endothelial monolayer integrity. To investigate the function of endothelial cell-secreted Ang[2] on endothelial integrity, we silenced Ang[2], Tie[2] or the related Tie[1] receptor in human dermal blood microvascular endothelial cells (BECs), which secrete endogenous Ang[2] stored in Weibel–Palade bodies[28]. These results suggest that b1-integrin in the cell periphery maintains the endothelial cortical actin cytoskeleton, and that Ang[2] promotes cytoskeletal changes via b1-integrin deposition in the central elongated adhesions, which support actin stress fibre formation. Av- and b3-integrin silencing resulted in disorganized cortical actin (Fig. 3e,f; Supplementary Fig. 3e), in line with previous reports implicating avb[3] in the stabilization of the cortical actin cytoskeleton in endothelial cells[38,39] Taken together, these data demonstrate that the cytoskeletal and junctional alterations induced by Tie[2] silencing in BECs are not dependent on av-integrins, and instead rely on b1-integrin function. To investigate Ang[2] signalling in the absence of Tie[2], we used the HeLa adenocarcinoma cell shIntegrin β1 shTie2+shIntegrin β1

80 Integrin β1

CHO-Tie2-ECD ***

Methods