Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling
Moritz Felcht, … , Sergij Goerdt, Hellmut G. Augustin
Moritz Felcht, … , Sergij Goerdt, Hellmut G. Augustin
Published May 15, 2012
Citation Information: J Clin Invest. 2012;122(6):1991-2005. https://doi.org/10.1172/JCI58832.
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Research Article Vascular biology

Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling

Abstract

Angiopoietin-2 (ANG-2) is a key regulator of angiogenesis that exerts context-dependent effects on ECs. ANG-2 binds the endothelial-specific receptor tyrosine kinase 2 (TIE2) and acts as a negative regulator of ANG-1/TIE2 signaling during angiogenesis, thereby controlling the responsiveness of ECs to exogenous cytokines. Recent data from tumors indicate that under certain conditions ANG-2 can also promote angiogenesis. However, the molecular mechanisms of dual ANG-2 functions are poorly understood. Here, we identify a model for the opposing roles of ANG-2 in angiogenesis. We found that angiogenesis-activated endothelium harbored a subpopulation of TIE2-negative ECs (TIE2lo). TIE2 expression was downregulated in angiogenic ECs, which abundantly expressed several integrins. ANG-2 bound to these integrins in TIE2lo ECs, subsequently inducing, in a TIE2-independent manner, phosphorylation of the integrin adaptor protein FAK, resulting in RAC1 activation, migration, and sprouting angiogenesis. Correspondingly, in vivo ANG-2 blockade interfered with integrin signaling and inhibited FAK phosphorylation and sprouting angiogenesis of TIE2lo ECs. These data establish a contextual model whereby differential TIE2 and integrin expression, binding, and activation control the role of ANG-2 in angiogenesis. The results of this study have immediate translational implications for the therapeutic exploitation of angiopoietin signaling.

Authors

Moritz Felcht, Robert Luck, Alexander Schering, Philipp Seidel, Kshitij Srivastava, Junhao Hu, Arne Bartol, Yvonne Kienast, Christiane Vettel, Elias K. Loos, Simone Kutschera, Susanne Bartels, Sila Appak, Eva Besemfelder, Dorothee Terhardt, Emmanouil Chavakis, Thomas Wieland, Christian Klein, Markus Thomas, Akiyoshi Uemura, Sergij Goerdt, Hellmut G. Augustin

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Figure 8

ANG-2–induced enhancement of VEGF sprouting and FAK (Tyr397) phosphorylation require αvβ3, αvβ5, and α5β1 integrins.

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ANG-2–induced enhancement of VEGF sprouting and FAK (Tyr397) phosphoryla...
(A) Spheroid sprouting assay with shRNA control ECs or lentivirally silenced TIE2 ECs in the presence of control IgG or the integrin antibody cocktail against αvβ3, αvβ5, and α5β1 (Integrin AB). Spheroids were stimulated with VEGF, ANG-2, or a combination of both, and the cumulative sprout length was quantified (*P < 0.05; n = 3). (B) Effect of ANG-2 on p-FAK (Tyr397) activation. Control and TIE2-silenced HUVECs were preincubated with the αvβ3, αvβ5, and α5β1 integrin antibody cocktail and adhered with or without Mn2+ to ANG-2–coated dishes (BSA, negative control; fibronectin, positive control). Blots of adherent and non-adherent cell lysates were probed for TIE2, p-FAK (Tyr397), and total FAK. Dotted boxes mark the effect of integrin blockage on ANG-2–induced p-FAK (Tyr397) (lane 10 versus 11) (ImageJ quantitation; n = 3; 1-tailed Student’s t test, *P < 0.05). (C) Spheroid sprouting was induced by ANG-2 and VEGF with IgG control or integrin blocking antibody cocktail in TIE2-silenced ECs. Spheroids were stained for p-FAK (Tyr397) and visualized together with lentiviral GFP by confocal microscopy. (D) The cornea pocket assay was performed with IgG control or αvβ3, αvβ5, and α5β1 antibody blockage. Angiogenesis was induced with ANG-2 and VEGF. The combination of ANG-2 and VEGF induced sprouting in the IgG control and FAK phosphorylation at Tyr397 (upper panel, arrows with asterisk). In contrast, integrin blockage inhibited sprouting and p-FAK (Tyr397) activation (arrow) (white dotted line marks corneal limbus). A higher-magnification image is shown in the inset. Scale bars: 30 μm.