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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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).
Integrated supplementary information
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.
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.
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.
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.
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.
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.
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.
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.
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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Scientific Reports (2015)