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. 2012 Jun;122(6):1991-2005.
doi: 10.1172/JCI58832. Epub 2012 May 15.

Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling

Moritz Felcht  1 , 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
Affiliations

Angiopoietin-2 differentially regulates angiogenesis through TIE2 and integrin signaling

Moritz Felcht et al. J Clin Invest. 2012 Jun.

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.

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Figures

Figure 1
Figure 1. ANG-2 blockage inhibits angiogenesis by interfering with remodeling of the stalk and phalanx cell vasculature as well as by inhibiting the sprouting tip cell EC phenotype.
(A) Newborn mice were injected intraperitoneally with 30 μg/pup neutralizing ANG-2 antibody (red bars throughout) or IgG control antibody (blue bars) at P1, P3, and P5. Mice were sacrificed at day 6, and the enucleated eyes were processed for retinal whole mount analysis and staining with FITC-labeled lectin (scale bars: 400 μm). (B–I) Quantitative analysis of the vasculature with Fiji analysis (see Supplemental Experimental Procedures) characterizing the total vessel area (scale bar: 400 μm) (B and C), the vessel density (D), the non-vascularized area (defined as an area that was more than 40 μm away from the next vessel [green]) (scale bars: 400 μm) (E and F), the junctional branch points per retina (G), the vessel segments per retina (H), and histogram of the branch length distribution (I). For B–H, *P < 0.05. (J–L) Higher-resolution images at the edge of the vascular plexus (red arrows, tip cells; yellow arrow, filopodia) demonstrating the pronounced tip cell phenotype–inhibiting effect of the ANG-2 blocking antibody with reduced total number of tip cells/retina (K) and fewer filopodia/tip cell (L). (M–O) ANG-2 blockage inhibited tumor growth of subcutaneously growing Colo205 xenografts after anti–ANG-2 treatment (*P < 0.05, mean ± SEM, n = 20) (M). Colo205 xenograft tumor sections were stained for CD34 and analyzed by high-resolution analysis to detect intratumoral endothelial tip cells (scale bars: 50 μm) (N). The number of intratumoral tip cells was significantly reduced in the ANG-2 antibody treatment group (*P < 0.05, mean ± SEM, n = 5) (O).
Figure 2
Figure 2. Tip cells have weak TIE2, but strong ANG-2 expression.
(A and B) TIE2 expression in lectin- and TIE2-double-stained retinas from adult mice (A) and postnatal mouse pups (B) (n = 3 each; arrows: TIE2-positive ECs; arrowheads: TIE2-negative ECs). (C) Specimen of human melanoma (n = 6) and healthy skin specimens (n = 3) were stained for CD34 and TIE2. The resting vasculature in the control specimen uniformly coexpressed TIE2 and CD34 (arrows, top row). In contrast, TIE2-positive (arrows) and TIE2-negative ECs (arrowheads) were detected in the vasculature of melanomas (bottom row). (D) Co-localization of CD34 and TIE2 in the spheroidal EC xenografting assay (30, 31). Lumenized vessels stained positive for TIE2 (arrow indicating yellow co-localization; left). ECs in non-lumenized vascular structures stained for CD34 but not for TIE2 (arrowhead, right). (E) Comparative TIE2 expression of migrating and confluent HUVECs (scratch wound assay; image shown in pseudocolors; n = 3). (F) Quantitative assessment of mean TIE2 expression in migrating and confluent ECs (*P < 0.05). (G) FACS analysis of confluent and subconfluent ECs for uPAR and TIE2 expression. (H) Quantitative assessment of the mean EC subpopulation of TIE2-negative and uPAR-positive ECs under non-permeating conditions. sub, subonfluent; con, confluent. (I) Abundant Ang2 mRNA expression in tip cells of the developing retina. Whole mount retinas were analyzed by in situ hybridization against Ang2, followed by immunoreactivity against collagen IV. Merged signals were pseudocolored using Adobe Photoshop CS software. Scale bars: A, 75 μm; B, 300 μm (insets, 50 μm); C, 75 μm; D, 75 μm; E, 100 μm (insets, 20 μm); I, 20 μm.
Figure 3
Figure 3. ANG-2 induces Rac activation and migration in TIE2lo ECs.
(A) Migration of control transduced (sh-Ctrl) and TIE2-silenced ECs (sh-TEK) cotransduced with control adenovirus (Ad-Ctrl) or adenoviral ANG-2 (Ad-ANG-2) in an 18-hour lateral scratch wound migration assay. The white lines denote the wound closure front of migrating ECs. (B) Quantification of lateral sheet migration shown in A (n = 3). The migration-stimulating effect was most pronounced when TIE2 was silenced and ANG-2 overexpressed at the same time. *P < 0.05. (C) Single-cell migration of control transduced ECs and TIE2-silenced ECs cotransduced with control adenovirus or adenoviral ANG-2. The starting point of individual cells was recorded (top left), and the cells were allowed to migrate for 24 hours (bottom left). The overall distance (dotted line) as well as the net distance (solid line) were recorded (top right). (D) Quantification of the net distance of the single-cell tracking assay shown in C. Migration of 10 cells per experimental group was expressed as net distance compared with Ad-Ctrl (*P < 0.05; n = 4). (E) Quantification of persistence (net distance divided by overall distance) of the single-cell tracking assay show in C. Persistence of 10 cells per experimental group was analyzed and expressed as relative persistence compared to Ad-Ctrl (*P < 0.05; n = 4). (F–I) Biochemical analysis of Rac1 activation (Western blot), with quantitative assessment of 7 independent experiments (mean ± SEM; *P < 0.05) (I). Two representative Western blots are shown (G and H). TIE2 silencing was monitored by Western blotting (F).
Figure 4
Figure 4. ANG-2 overexpression enhances vascular network formation of TIE2lo ECs.
(A) Vessel network formation of control transduced and TIE2-silenced ECs cotransduced with control lentivirus (p-Ctrl) or lentiviral ANG-2 (p-ANG-2) in the Matrigel xenografting assay was analyzed as described previously (30, 31). Sections (50 μm) were stained for CD31 and analyzed by Fiji software for mean vessel density (B), branch points (C), junctions (D), and vascular tips (E). Red bars indicate sh-Ctrl/p-Ctrl; yellow bars, p-ANG-2/sh-Ctrl; blue bars, p-Ctrl/sh-TEK; green bars p-ANG-2/sh-TEK ECs (*P < 0.05; mean ± SEM; n ≥ 3). Scale bars: 70 μm.
Figure 5
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.
(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).
Figure 6
Figure 6. ANG-2 binds αvβ3, αvβ5, and α5β1 integrins.
(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.
Figure 7
Figure 7. ANG-2 binds to αvβ3, αvβ5, and α5β1 integrins with lower affinity than TIE2 receptor.
(A) ELISA quantification of ANG-2 adhesion to TIE2 or integrins at physiological pH or in an acidic environment. ANG-2 bound in an acidic environment with significantly higher affinity to TIE2 compared with the integrin heterodimers (*P < 0.05; n = 3). (B–D) HUVEC monolayers were stimulated with Mn2+ or ANG-2. The integrin conformation was studied with β1 integrin active conformation antibodies (HUTS-21, 9EGF) (40, 41). The mean intensity was determined and the relative conformation expression quantified (n = 3). In contrast to Mn2+ stimulation, ANG-2 did not induce conformational changes in β1 integrins. (E) Effect of Mn2+ on HUVEC adhesion to immobilized ANG-2. HUVECs adhered to ANG-2–coated plates with or without Mn2+. Adherent cells were visualized with crystal violet. (F) Competition binding of ANG-2 and RGD-containing proteins. After preincubation of ECs with ANG-2, cells adhered to plates coated with fibronectin (FN) or vitronectin (VN). Adherent cells were visualized with crystal violet. Preincubation with ANG-2 did not reduce binding to RGD-containing FN or VN but enhanced HUVEC adhesion to FN (*P < 0.05; n = 3). (G) Antibody blocking of control transduced and TIE2-silenced HUVEC adhesion to ANG-2. HUVECs were preincubated with the indicated integrin heterodimer antibodies (cocktail: combination of αvβ3, αvβ5, and α5β1 integrin antibodies), followed by adhesion to ANG-2. Adherent cells were visualized with crystal violet. Antibodies against αvβ3, α5β1, and the integrin cocktail inhibited HUVEC adhesion to the ANG-2 matrix to baseline levels in TIE2lo ECs (*P < 0.05; n = 3).
Figure 8
Figure 8. ANG-2–induced enhancement of VEGF sprouting and FAK (Tyr397) phosphorylation require αvβ3, αvβ5, and α5β1 integrins.
(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.
Figure 9
Figure 9. Model of the contextual bifunctional effects of ANG-2 during angiogenesis.
(A) Quiescent resting ECs express TIE2. EC activation leads to secretion of Weibel-Palade body–stored ANG-2 and strong transcriptional ANG-2 upregulation. ANG-2 interferes with ANG-1–induced TIE2 activation to destabilize the resting EC monolayer and primes it to respond to exogenous cytokines. Endothelial destabilization is at least in part mediated by ANG-2/TIE2/integrin complex formation, which subsequently leads to FAK phosphorylation at Ser910, as well as integrin internalization and degradation (14). Thus, among the effects of ANG-2, the antagonistic mode of action predominates in resting ECs. (B) Angiogenic activation has a negative effect on EC TIE2 expression. As a result, angiogenic tip cells are TIE2 low or negative, whereas remodeling stalk and phalanx cells express TIE2. Conversely, expression of the integrins αvβ3, αvβ5, and α5β1 is upregulated on angiogenic ECs. In the absence of TIE2, ANG-2 directly binds and activates integrins, which are in their conformationally active state in angiogenically activated ECs. Integrin activation induces FAK phosphorylation at Tyr397, Rac1 activation, and EC migration. As a result, the combination of differential receptor expression and affinity controls the net outcome of ANG-2 signaling. This allows ANG-2 to control different steps of the angiogenic cascade in a bifunctional manner.
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References

    1. Augustin HG, Koh GY, Thurston G, Alitalo K. Control of vascular morphogenesis and homeostasis through the angiopoietin-Tie system. Nat Rev Mol Cell Biol. 2009;10(3):165–177. - PubMed
    1. Gale NW, et al. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev Cell. 2002;3(3):411–423. doi: 10.1016/S1534-5807(02)00217-4. - DOI - PubMed
    1. Fiedler U, et al. Angiopoietin-2 sensitizes endothelial cells to TNF-alpha and has a crucial role in the induction of inflammation. Nat Med. 2006;12(2):235–239. doi: 10.1038/nm1351. - DOI - PubMed
    1. Koh YJ, et al. Double antiangiogenic protein, DAAP, targeting VEGF-A and angiopoietins in tumor angiogenesis, metastasis, and vascular leakage. Cancer Cell. 2010;18(2):171–184. doi: 10.1016/j.ccr.2010.07.001. - DOI - PubMed
    1. Huang H, Bhat A, Woodnutt G, Lappe R. Targeting the ANGPT-TIE2 pathway in malignancy. Nat Rev Cancer. 2010;10(8):575–585. doi: 10.1038/nrc2894. - DOI - PubMed

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