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ADAMTS-2 functions as anti-angiogenic and anti-tumoral molecule independently of its catalytic activity

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Abstract

ADAMTS-2 is a metalloproteinase that plays a key role in the processing of fibrillar procollagen precursors into mature collagen molecules by excising the amino-propeptide. We demonstrate that recombinant ADAMTS-2 is also able to reduce proliferation of endothelial cells, and to induce their retraction and detachment from the substrate resulting in apoptosis. Dephosphorylation of Erk1/2 and MLC largely precedes the ADAMTS-2 induced morphological alterations. In 3-D culture models, ADAMTS-2 strongly reduced branching of capillary-like structures formed by endothelial cells and their long-term maintenance and inhibited vessels formation in embryoid bodies (EB). Growth and vascularization of tumors formed in nude mice by HEK 293-EBNA cells expressing ADAMTS-2 were drastically reduced. A similar anti-tumoral activity was observed when using cells expressing recombinant deleted forms of ADAMTS-2, including catalytically inactive enzyme. Nucleolin, a nuclear protein also found to be associated with the cell membrane, was identified as a potential receptor mediating the antiangiogenic properties of ADAMTS-2.

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Abbreviations

EB:

Embryoid body

EBM:

Endothelial basal medium

ES cell:

Embryonic stem cell

HEK 293-EBNA cell:

Human embryonic kidney 293-Epstein Barr nuclear antigen cell

HMEC:

Human microvascular endothelial cell

HMVEC:

Human dermal microvascular endothelial cell

HSF:

Human skin fibroblasts

HSMC:

Human smooth muscle cell

HSPG::

Heparan sulfate proteoglycan

HUVEC:

Human umbilical vein endothelial cell

MLC:

Myosin light chain

mTS2:

Catalytically inactive ADAMTS-2

PAK:

p21 Activated kinase

PECAM:

Platelet endothelial cell adhesion molecule: CD31

ROCK:

Rho kinase

TSR1:

Thrombospondin repeat type 1

TS-2:

ADAMTS-2

wtTS2:

Wild-type ADAMTS-2

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Acknowledgments

This work was supported by grants from the Belgian Fonds de la Recherche Scientifique Médicale (Grant nos. 3.4362.03 and 3.4387.05), the Belgian Fonds National de la Recherche Scientifique, the “Foundation against Cancer”, the “Fondation Léon Fredericq” (University of Liège), the “Centre Anticancéreux près l’Université de Liège” and the “Région Wallonne” (NeoAngio, grant no. 616476). We thank the “Proteomic” (M.-A. Meuwis) and “Imaging and Flow cytometry” (Sandra Ormenese) platforms of GIGA-R (University of Liège) and Dr. Prockop (Texas A&M Health Science Center, USA) for the kind gift of ADAMTS-2 −/− mice.

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Authors and Affiliations

  1. Laboratory of Connective Tissues Biology, GIGA-R, Tour de Pathologie, B23/3, 4000, Sart Tilman, Belgium

    J. Dubail, F. Kesteloot, C. Deroanne, C. Lapière, B. Nusgens & A. Colige

  2. Laboratory of Plant Cellular Biology, Sart Tilman, Belgium

    P. Motte

  3. Laboratory of Development and Tumor Biology, University of Liège, Sart Tilman, Belgium

    V. Lambert

  4. Department of Ophthalmology, University Hospital, Sart Tilman, Belgium

    J.-M. Rakic

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  1. J. Dubail
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Correspondence to A. Colige.

Electronic supplementary material

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18_2010_431_MOESM1_ESM.tif

Figure S1: ADAMTS-2 alters the morphology of endothelial cells cultured on different substrates. HUVEC or HMEC were seeded on gelatin (k, m, o, q) or fibronectin (l, n, p, r) coats for 3 hours in serum free EBM and cultured for 16 hours in control medium (k, l, o, p) or in the presence of ADAMTS-2 at 5 µg/ml (m, n, q, r). Bar = 100 µm. (TIFF 4209 kb)

18_2010_431_MOESM2_ESM.tif

Figure S2: Merge pictures of Fig. 2 a-b and c-d. HUVEC were seeded on fibronectin coat for 3 hours in serum-free EBM and cultured for 16 hours in control medium or in medium supplemented with ADAMTS-2 at 5 µg/ml. Cells were fixed, permeabilized with 0.1% Triton X-100, and stained to visualize actin stress fibers (FITC-Phalloidin; green) and focal adhesions (anti-vinculin mAb and TRITC-conjugated secondary antibody; red). Nuclei were stained with DAPI (blue). Bar = 50 µm. Merge picture (TIFF 3178 kb)

18_2010_431_MOESM3_ESM.tif

Figure S3: Identification of nucleolin as a binding partner for ADAMTS-2. Plasma membrane extracts of HMEC were prepared and applied to a column containing immobilized ADAMTS-2 or the uncoupled resin as control. Bound proteins identified by by micro high performance liquid chromatography-electrospray ionization-trap (µHPLC-ESI-Trap) were eluted at 0.5 M NaCl, separated by SDS-PAGE and analysed by Western blotting using antibody directed against nucleolin. (TIFF 413 kb)

18_2010_431_MOESM4_ESM.tif

Figure S4: Choroidal neovascularisation in wild type and ADAMTS-2 -/- mice. LASER impacts were used to injure, as previously described [46], the choroids of wild type (WT) or homozygous ADAMTS-2 deficient mice (ADAMTS-2-/-) [23] in order to induce an angiogenic response. After 14 days, mice were injected with dextran coupled to fluorescein to visualize blood vessels by fluorescence imaging. Flats mounts of the entire choroids were visualized by confocal microscopy (A). Quantification of the angiogenic response was performed by digital image analysis of the green spots surface (n = 8) (B). Bar = 80 µm. (*: p<0.05) (TIFF 954 kb)

Supplementary material 5 (DOC 41 kb)

18_2010_431_MOESM6_ESM.mpg

Video 1: Morphology of endothelial cells in control conditions. HUVEC were seeded for 5 hours in EBM supplemented with 0.5% FBS and cultured for 20 hours in control medium. Images were recorded every 5 minutes for 20 hours. Videos were performed with 8 frames/second display rate. (MPG 2898 kb)

18_2010_431_MOESM7_ESM.mpg

Video 2: ADAMTS-2 rapidly induces modifications of endothelial cells morphology. HUVEC were seeded for 5 hours in EBM supplemented with 0.5% FBS and cultured for 20 hours in presence of ADAMTS-2 at 5 μg/ml. Images were recorded every 5 minutes for 20 hours. Videos were performed with 8 frames/second display rate. (MPG 2886 kb)

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Dubail, J., Kesteloot, F., Deroanne, C. et al. ADAMTS-2 functions as anti-angiogenic and anti-tumoral molecule independently of its catalytic activity. Cell. Mol. Life Sci. 67, 4213–4232 (2010). https://doi.org/10.1007/s00018-010-0431-6

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