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 5

ANG-2 induces in TIE2hi ECs phosphorylation of the integrin adaptor protein FAK at Ser910 and in TIE2lo ECs phosphorylation of FAK at Tyr397.

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ANG-2 induces in TIE2hi ECs phosphorylation of the integrin adaptor prot...
(A) Confluent EC monolayers were stimulated with ANG-2. Blots of total cell lysates were probed for FAK phosphorylation at Ser910 (pSer910), Tyr397 (pTyr397), total FAK, and actin or tubulin. (B) ImageJ ( http://rsb.info.nih.gov/ij/) quantification of FAK phosphorylation at Ser910 or Tyr397 (n ≥ 3; normalized to tubulin; 1-tailed Student’s t test, *P < 0.05). (C–F) FAK phosphorylation at Tyr397 of control transduced and TIE2-silenced ECs cotransduced with control lentivirus or lentiviral ANG-2 was analyzed in the Matrigel xenografting assay. Sections (50 μm) were stained for CD31 and p-FAK (Tyr397) (Imaris 7.2.3 visualization with pseudocolors). Scale bar: 100 μm. (G and H) Cornea pocket assay experiments were performed with double implantation of ANG-2 and subcritical doses of VEGF (see Supplemental Figure 8 for an overview of a representative cornea). VEGF-induced sprouting was enhanced by ANG-2 and was accompanied by p-FAK (Tyr397) activity in tip cells (positive: arrows with asterisks; negative: arrow without asterisk). The white dotted line marks the limbus of the cornea. Scale bars: 40 μm. (I–L) Postnatal mouse pups were systemically treated with ANG-2 blocking antibody (see Supplemental Figure 1A for details), followed by staining for CD31 and p-FAK (Tyr397). Images were taken with z-stack and tilt confocal microscopy. The retinal endothelium strongly expressed p-FAK (Tyr397) (see Supplemental Figure 8B and Supplemental Video 3). Images were assessed with the colocalization function of Imaris 7.2.3. Scale bars: 300 μm for overview (I and K) and 50 μm for higher-magnification images (J and L).