Article | Published:

Endothelial podosome rosettes regulate vascular branching in tumour angiogenesis

Nature Cell Biology volume 16, pages 931941 (2014) | Download Citation

Abstract

The mechanism by which angiogenic endothelial cells break the physical barrier of the vascular basement membrane and consequently sprout to form new vessels in mature tissues is unclear. Here, we show that the angiogenic endothelium is characterized by the presence of functional podosome rosettes. These extracellular-matrix-degrading and adhesive structures are precursors of de novo branching points and represent a key feature in the formation of new blood vessels. VEGF-A stimulation induces the formation of endothelial podosome rosettes by upregulating integrin α6β1. In contrast, the binding of α6β1 integrin to the laminin of the vascular basement membrane impairs the formation of podosome rosettes by restricting α6β1 integrin to focal adhesions and hampering its translocation to podosomes. Using an ex vivo sprouting angiogenesis assay, transgenic and knockout mouse models and human tumour sample analysis, we provide evidence that endothelial podosome rosettes control blood vessel branching and are critical regulators of pathological angiogenesis.

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Acknowledgements

A special thank you to E. Georges-Labouesse (CNRS/INSERM/ULP, Illkirch, France), who recently passed away, for kindly providing Tie2-dependent integrin α6 KO mice. We thank P. C. Marchisio (San Raffaele Scientific Institute, Milano, Italy) for discussion and insightful suggestions on the manuscript; D. R. Sherwood (Duke University Medical Center, Durham, USA) for critical reading of the manuscript; K. Tryggvason (Karolinska Institutet, Stockholm, Sweden) for providing laminin α4 null mice; R. Wedlich-Söldner (Max-Planck Institute of Biochemistry, Martinsried, Germany) and L. M. Machesky (Beatson Institute for Cancer Research, Glasgow, UK) for providing breeding pairs for the LifeAct–EGFP mouse colony and reagents; R. Falcioni (National Cancer Institute ‘Regina Elena’, Rome, Italy) for critical reading and reagents; E. De Luca and M. Gai (MBC, Torino, Italy) for their assistance in multiphoton microscopy; Y. Boucher and C. Smith (HMS, Boston, USA) for assistance in immunohistochemistry on human tissues; and E. Giraudo and F. Maione (IRCC, Candiolo, Italy) for their help in the treatment of RipTag2 mice. This work was supported by Associazione Italiana per la Ricerca sul Cancro (AIRC) investigator grants IG (10133, F.B.; 14635, L.P.; 13016 G. Serini) and fellowships (13604 G. Seano; 15026 P.A.G.); AIRC 5x1000 (12182); Converging Technologies Program, grant: ‘Photonic Biosensors for Early Cancer Diagnostics’; Technological Platforms for Biotechnology: grant DRUIDI; Fondazione Cassa di Risparmio Torino (CRT); Fondazione Piemontese per la Ricerca sul Cancro-ONLUS (Intramural Grant 5x1000 2008) (L.P.); Fondo Investimenti per la Ricerca di Base RBAP11BYNP (Newton) (F.B. and L.P.); University of Torino-Compagnia di San Paolo: RETHE grant (F.B.); GeneRNet grant (L.P.); P01 CA080124/CA/NCI NIH HHS/United States (R.K.J.); The ‘Fondazione T. & L. de Beaumont Bonelli’ and the Girardi Family (G. Seano).

Author information

Author notes

    • Claire Bouvard
    •  & Roberto Sessa

    Present addresses: The Scripps Research Institute, La Jolla, California 92037, USA (C.B.); Center for Eye Disease and Development, University of California, Berkeley, California 94720, USA (R.S.).

Affiliations

  1. Department of Oncology, University of Torino, Turin 10100, Italy

    • Giorgio Seano
    • , Giulia Chiaverina
    • , Paolo Armando Gagliardi
    • , Laura di Blasio
    • , Alberto Puliafito
    • , Roberto Sessa
    • , Guido Serini
    • , Federico Bussolino
    •  & Luca Primo
  2. Candiolo Cancer Institute-FPO, IRCCS, Candiolo 10060, Italy

    • Giorgio Seano
    • , Giulia Chiaverina
    • , Paolo Armando Gagliardi
    • , Laura di Blasio
    • , Alberto Puliafito
    • , Roberto Sessa
    • , Guido Serini
    • , Federico Bussolino
    •  & Luca Primo
  3. Edwin L. Steele Laboratory for Tumor Biology, Harvard Medical School, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

    • Giorgio Seano
    •  & Rakesh K. Jain
  4. UMR-S 765, Université Paris Descartes, Sorbonne Paris Cité, Paris 75006, France

    • Claire Bouvard
  5. Department of Molecular Biotechnology and Health Sciences, University of Torino, Molecular Biotechnology Center, Turin 10124, Italy

    • Guido Tarone
  6. Institute of Physiological Chemistry and Pathobiochemistry, Muenster University, Muenster 48149, Germany

    • Lydia Sorokin
  7. UMR-S 970, Université Paris Descartes, Sorbonne Paris Cité, Paris 75006, France

    • Dominique Helley

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Contributions

G. Seano and L.P. conceived the idea and wrote the manuscript; G. Seano, G.C., P.A.G., A.P. and L.d.B. performed the experiments and analysed the data; L.S. provided LAMA4 mARs; C.B. and D.H. provided endothelial α6 null mARs, B16F10 tumours and ischaemic tissues; R.K.J. provided human samples; G. Serini, G.T., R.K.J. and F.B. analysed and discussed the data; all authors reviewed and approved the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Giorgio Seano or Luca Primo.

Integrated supplementary information

Supplementary information

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  1. 1.

    Supplementary Information

    Supplementary Information

Videos

  1. 1.

    Actin dynamics in endothelial podosome rosettes formation.

    Time-lapse microscopy of LifeAct-RFP localization in EC treated with PMA for the indicated time. Pseudocolors: TIRF in green and EPI in red. EC were seeded on gelatin-coated glass-bottom dishes. Scale bar, 10 μm.

  2. 2.

    Integrin α6 dynamics in adhesive structures during PMA treatment in EC seeded on laminin-rich substrates or not.

    Time-lapse TIRF microscopy of LifeAct-RFP (red) and α6-GFP (green) localization in EC treated with PMA for the indicated time. EC were seeded on glass-bottom dishes coated with gelatin plus laminin at indicated concentrations. Scale bar, 15 μm.

  3. 3.

    Focal adhesions and podosome rosettes dynamics during PMA treatment.

    Time-lapse TIRF microscopy of vinculin-RFP (black) localization in EC. EC were cultured in basal medium and then treated with basal medium plus PMA at the indicated time. EC were seeded on gelatin-coated glass-bottom dishes. Scale bar, 20 μm.

  4. 4.

    3D reconstruction of endothelial podosome rosettes in angiogenic outgrowths.

    3D reconstruction of angiogenic outgrowth from mAR into collagen gel. mAR were stimulated with VEGF-A and FGF-2 for 7 days, then fixed and immunostained. Isosurface of F-actin staining was coloured in gray and endothelial rosettes – colocalization of cortactin and F-actin – in red.

  5. 5.

    Endothelial podosome rosettes in angiogenic outgrowth from LifeAct-EGFP mAR.

    Xyz-section of time-lapse 2-photon microscopy of angiogenic outgrowths from LifeAct-EGFP mAR, stimulated with VEGF-A and FGF-2. In the video the formation of a 5-6 μm-diameter rosette is evident, followed by a cell protrusion of 14-16 μm of length. Top-left panel is the x-plane, top-right is the z-plane, bottom-left is the y-plane and bottom-right is the image. Scale bar, 20 μm.

  6. 6.

    Branching from endothelial rosettes in LifeAct-EGFP mAR.

    Time-lapse 2-photon microscopy of angiogenic outgrowths from LifeAct-EGFP mAR, stimulated with VEGF-A and FGF-2. Inset, 3D reconstruction of endothelial podosome rosette of the same video. Scale bar, 50 μm.

  7. 7.

    Dynamical analysis of vessel branching in endothelial ITGA6 KO mAR.

    Time-lapse phase-contrast microscopy of angiogenic outgrowths from mAR. mAR from WT (α6fl/fl-Tie2Cre-) or endothelial α6 KO (α6fl/fl-Tie2Cre+) mice were stimulated with VEGF-A and FGF-2. Scale bar, 70 μm.

  8. 8.

    Dynamical analysis of vessel branching in Lama4−/− KO mAR.

    Time-lapse phase-contrast microscopy of angiogenic outgrowths from mAR. mAR from WT or Laminin α4 null (LAMA4 mAR) mice were stimulated with VEGF-A and FGF-2. Scale bar, 70 μm.

  9. 9.

    Dynamical analysis of vessel branching in mAR into laminin-rich matrices.

    Time-lapse phase-contrast microscopy of angiogenic outgrowths from mAR into type-I-collagen gel with or without 20 μg/ml of laminin addition. mARs were stimulated with VEGF-A and FGF-2. Scale bar, 70 μm.

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DOI

https://doi.org/10.1038/ncb3036

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