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New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding, cellular signaling and vessels formation

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Abstract

VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1–4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF111a, VEGF111b, VEGF121a, VEGF121b, VEGF155a, VEGF155b, VEGF165a, VEGF165b) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF165b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF111b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant.

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Acknowledgments

The scientific advice of Professor B. Nusgens, the technical assistance of A. Hoffmann, A. Heyeres, F. Olivier and L. Duwez and the informatic skill of N. Garbacki were greatly appreciated. We thank the Genotranscriptomics and Proteomics Platforms of the GIGA (University of Liège). This work was supported by the “Belgian Foundation against Cancer, Nonprofit Organization” (197-2008-FCC-VEGF111), the “Région Wallonne” (DGO6, n° 816865), Skin Cancer Research Fund (ScaRF), INSERM, University Paris 13 and FP7 Nanoantenna Project from EU.

Conflict of interest

Prof Bates is an inventor on patents describing the potential uses of VEGF165b. The other authors declare no conflict of interest.

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Authors and Affiliations

  1. Laboratory of Connective Tissues Biology, GIGA-R, University of Liège, Avenue de l’Hôpital 3, 4000, Liège, Belgium

    Romain Delcombel, Lauriane Janssen, Céline Hendricks, Christophe Deroanne, Charles Lambert & Alain Colige

  2. EA4222, Université Paris 13, Sorbonne Paris Cité, 74 rue Marcel Cachin, 93000, Bobigny, France

    Roger Vassy & Anna Starzec

  3. Microvascular Research Laboratories, School of Physiology and Pharmacology, University of Bristol, Preclinical Veterinary Sciences Building, Southwell Street, Bristol, BS2 8EJ, UK

    Melissa Gammons & David Bates

  4. Inserm U698, Université Paris 13, Sorbonne Paris Cité, 74 rue Marcel Cachin, 93000, Bobigny, France

    Oualid Haddad, Benjamin Richard & Didier Letourneur

  5. Department of Cardiology and Angiology, University Hospital of Münster, Albert-Schweitzer-Campus 1, A1, 48149, Münster, Germany

    Johannes Waltenberger

  6. Laboratory of Tumour and Development Biology, GIGA-R, University of Liège, Avenue de l’Hôpital 3, 4000, Liège, Belgium

    Nor Eddine Sounni & Agnès Noël

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  1. Romain Delcombel
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10456_2012_9320_MOESM1_ESM.tif

Supplemental Fig. 1 Binding of VEGF variants to Heparin. Binding of VEGF variants (200 nM) to heparin was measured by surface plasmon resonance. (a) HBS-EP buffer, (b) VEGF111a, (c) VEGF121a, (d) VEGF155a, (e) VEGF165a and (f) VEGF165b were injected (black arrow) for ~ 250 s followed by injection of HBS-EP buffer (open arrow) in order to visualize the dissociation rate. RU: Response in arbitrary units (TIFF 9789 kb)

10456_2012_9320_MOESM2_ESM.tif

Supplemental Fig 2 Effect of A7R and R8R on VEGFR2-VEGF-NRP1-H complex formation. (a-c) The formation of the VEGFR2-VEGF-NRP1-H complex was measured by surface plasmon resonance. VEGF-R2 was coated on the sensorchip and successive injections were made as indicated on the drawings. (d) Quantification of the response induced by the addition of NRP1 + H in the absence or presence of mimetic peptides (A7R and R8R). Results are expressed in percentage of values recorded in absence of peptide. b: HBS-EP buffer; 165a: VEGF165a; NRP1: neuropilin-1; H: heparin; A7R: peptide mimetic of E8a-encoded sequence; R8R: peptide mimetic of E5-encoded sequence (TIFF 9407 kb)

10456_2012_9320_MOESM3_ESM.tif

Supplemental Fig. 3 Time-course experiment evaluating the effects of VEGF variants on ERK1/2 phosphorylation in endothelial cells. PAEC-R2 (a) and HUVEC (b) were treated with 1nM of VEGF variants for 0, 1, 5, 15 and 30 min in serum free medium. Protein extracts were then analyzed by western blot using ERK1/2 (ERK) and phospho-ERK1/2 (P-ERK) specific antibodies (TIFF 29455 kb)

10456_2012_9320_MOESM4_ESM.tif

Supplemental Fig. 4 Effect of increasing concentrations of VEGF variants on ERK1/2 phosphorylation in endothelial cells. PAEC-R2 (a, c, e, g) and HUVEC (b, d, f, h) were incubated for 10 min in serum free medium containing 0.1nM (a, b), 0.3nM (c, d), 1nM (e, f) or 3nM (g, h) of the different VEGF variants. Protein extracts were then analyzed by western blot using antibodies against ERK1/2 (ERK) or phospho-ERK1/2 (P-ERK) (TIFF 32326 kb)

10456_2012_9320_MOESM5_ESM.tif

Supplemental Fig 5 Effect of R8R on VEGF-induced signaling in vitro. PAEC-R2 (a) and PAEC-R2-NRP1 (b) were treated for 10’ at 37°c with 1nM of VEGF111a or with VEGF165a in the absence or presence of R8R (300 μM). Protein extracts were then analyzed by Western blotting using VEGF-R2 (R2 tot) and phospho-VEGFR2 (P-R2) specific antibodies (c, d). Calculated P-R2/R2 ratio of in PAEC-R2 (c) and PAEC-R2-NRP1 (d). HUVECs proliferation was measured by WST-1 assay in absence of VEGF, in the presence of VEGF111a, in the presence of VEGF165a (250 pM), or in the presence of both VEGF165a (250 pM) and R8R (300 μM) (e). Only statistics related to the effects of R8R are reported. **p < 0.01, ***p < 0.001 (unpaired t test) (TIFF 9482 kb)

10456_2012_9320_MOESM6_ESM.tif

Supplemental Fig. 6 Quantification of the effects of VEGF variants in tumour angiogenesis using HEK293 cells expressing the various VEGF isoforms. Two millions HEK293 cells, either control or overexpressing VEGF variants were mixed with Matrigel and injected in the flank of nude mice. (a) The VEGF mRNA levels (upper panel) were measured by RT-PCR using primers P1 and P2 (see Table 1) that enable the amplification of all the VEGF variants with production of a single amplicon. The 28S rRNA was measured in parallel to normalize the quantities of RNA input in the reactions. * indicate the amplicon formed from an internal standard co-amplified with 28S rRNA to take into account potential variations of the PCR efficiency. Ratios of the CD31 stained surfaces in the tumour (b) or the adjacent skin (c) were quantified. * p < 0.05, ** p < 0.01, *** p < 0.001 (unpaired t test) (TIFF 27515 kb)

10456_2012_9320_MOESM7_ESM.tif

Supplemental Fig. 7 Immunolocalization of VEGF in tumours expressing the various VEGF isoforms. (a-i) VEGF immunostaining on paraffin sections showing the tumours; scale bar = 100 μm (TIFF 38925 kb)

10456_2012_9320_MOESM8_ESM.tif

Supplemental Fig. 8 Effects on tumour angiogenesis of co-expressing VEGF 165 a and VEGF 165 b. Two millions HEK293 cells were mixed with Matrigel and injected in the flank of nude mice. In addition to control cells, two ratio of HEK293 cells expressing VEGF165a and VEGF165b were evaluated, either 1 × 106 each (165a + 165b) or 4 × 105 expressing VEGF165a and 1.6 × 106 expressing VEGF165b (165a + 4x165b). See Fig 6 for comparison with cells expressing only VEGF165a or VEGF165b. (a) Macroscopic view of the tumours obtained in each group. Control (n = 5), 165a + 165b (n = 4), 165a + 4x165b (n = 5). (b) CD31 immunostaining on paraffin sections showing the tumour (T) and the adjacent skin (S) at two levels of magnification (scale bar = 100 μm). Black lined rectangles delineate the areas of the sections represented in (c) at higher magnification (scale bar = 25 μm) (TIFF 30269 kb)

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Delcombel, R., Janssen, L., Vassy, R. et al. New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding, cellular signaling and vessels formation. Angiogenesis 16, 353–371 (2013). https://doi.org/10.1007/s10456-012-9320-y

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