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 6

ANG-2 binds αvβ3, αvβ5, and α5β1 integrins.

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ANG-2 binds αvβ3, αvβ5, and α5β1 integrins. (A) Antibody blocking of H...
(A) Antibody blocking of HUVEC adhesion to an immobilized ANG-2 matrix. HUVECs were preincubated with the indicated antibodies to integrin monomers and heterodimers (Cocktail: combination of αvβ3, αvβ5, and α5β1 integrin antibodies), followed by adhesion to an ANG-2 matrix for 40 minutes. Non-adherent cells were removed, and adherent cells were visualized with crystal violet. Color intensities were measured at 550 nm. Antibodies against αvβ3, α5β1, and β1 integrin led to a significant inhibition of HUVEC adhesion to the ANG-2 matrix. Two different β1 antibodies achieved similar results (*P < 0.05 versus IgG; n = 4). (B and C) Control transduced ECs and TIE2-silenced ECs cotransduced with control lentivirus or lentiviral ANG-2 were grown to confluence. Immunoprecipitation for αvβ5 and α5β1 or control IgG was performed, followed by SDS-PAGE and detection of ANG-2 by Western blot analysis (upper blots). The membranes were stripped and probed for expression of the integrin monomer (lower blots). The intensity was measured by ImageJ, and the mean of at least 3 independent experiments was calculated. ND, not determined. (D–F) Interaction of ANG-2 with αvβ5 (D), α5β1 (E), and αvβ3 (F) in a cell-free co-immunoprecipitation assay. Samples were immunoprecipitated with the indicated integrin antibodies, and co-immunoprecipitation of ANG-2 was probed by Western blotting.