VEGFR1 and VEGFR2 involvement in extracellular galectin-1- and galectin-3-induced angiogenesis

Abstract

Aim: Accumulating evidence suggests that extracellular galectin-1 and galectin-3 promote angiogenesis. Increased expression of galectin-1 and/or galectin-3 has been reported to be associated with tumour progression. Thus, it is critical to identify their influence on angiogenesis.

Methods: We examined the individual and combined effects of galectin-1 and galectin-3 on endothelial cell (EC) growth and tube formation using two EC lines, EA.hy926 and HUVEC. The activation of vascular endothelial growth factor receptors (VEGFR1 and VEGFR2) was determined by ELISA and Western blots. We evaluated the VEGFR1 and VEGFR2 levels in endosomes by proximity ligation assay.

Results: We observed different responses to exogenous galectins depending on the EC line. An enhanced effect on EA.hy926 cell growth and tube formation was observed when both galectins were added together. Focusing on this enhanced effect, we observed that together galectins induced the phosphorylation of both VEGFR1 and VEGFR2, whereas galectin-1 and -3 alone induced VEGFR2 phosphorylation only. In the same way, the addition of a blocking VEGFR1 antibody completely abolished the increase in tube formation induced by the combined addition of both galectins. In contrast, the addition of a blocking VEGFR2 antibody only partially inhibited this effect. Finally, the addition of both galectins induced a decrease in the VEGFR1 and VEGFR2 endocytic pools, with a significantly enhanced effect on the VEGFR1 endocytic pool. These results suggest that the combined action of galectin-1 and galectin-3 has an enhanced effect on angiogenesis via VEGFR1 activation, which could be related to a decrease in receptor endocytosis.

Figure 1. Modulation of cell growth by exogenous galectins.

The effects of exogenous galectins on the growth of EA.hy926 (A) and HUVEC (B) cells were evaluated by the MTT assay. (A) The MTT conversion in EA.hy926 cells was measured one day after galectin stimulation at the indicated concentrations. (B) The MTT conversion in HUVECs was measured two days after galectin stimulation at the indicated concentrations. Each condition was tested with six replicates. The data (mean +/− SEM) are shown as relative values compared with the control (no galectin addition), and significant differences are indicated (* p<0.05, ** p<0.01 and *** p<0.001).

Figure 2. Modulation of tube formation by exogenous galectins.

EA.hy926 (A, C, E) and HUVEC (B, D, F) cells were suspended in complete medium in the presence or absence of galectins at the indicated concentrations and seeded on top of matrigel layers. Representative images obtained at 22 h for EA.hy926 (A) and 6 h for HUVEC (B) are shown. Tube formation was quantified by measuring the total length of the tube network (C, D) or by counting branching point (E, F) in EA.hy926 cells (C, E) and HUVECs (D, F). The data (mean +/− SEM) are shown as relative values compared with the control (no galectin addition), and significant differences are indicated (* p<0.05, ** p<0.01 and *** p<0.001). Scale bar: 300 µm.

Figure 3. Effects of exogenous galectins on VEGFR activation and involvement of VEGFRs in galectin-induced tube formation.

(A–D) Determination of VEGFR1 (A, C) and VEGFR2 (B, D) phosphorylation levels following a 5-min stimulation of EA.hy926 cells with galectin-1, galectin-3 or both galectins (1 µg/ml each) by ELISA (A, B) and Western blots (C, D). For ELISAs, the data (mean +/− SEM) are shown as relative values compared with the control (no galectin addition), and significant differences are indicated (* p<0.05, ** p<0.01 and *** p<0.001). Quantification of Western blots was done using ImageJ (see Materials and Methods). (E–H) EA.hy926 cells were suspended in complete medium in the presence or absence of galectins (1 µg/ml each) and blocking VEGFR1 Ab (5 µg/ml) or control IgG (5 µg/ml) (E, G) or blocking VEGFR2 Ab (50 ng/ml) or control IgG (50 ng/ml) (F, H) and seeded on top of matrigel layers. Tube formation was quantified by measuring the total length of the tube network (E–F) or counting branching points (G–H). The data (mean +/− SEM) are shown as relative values compared with the control (without the addition of galectins or an inhibitor). Significant differences are indicated on horizontal arrows (the same galectin-related conditions were compared in the absence or presence of a blocking Ab using the Mann-Whitney test. * p<0.05, ** p<0.01 and *** p<0.001).

Figure 4. Galectin-induced activation of ERK1/2 and Hsp27.

Determination of ERK1/2 (A, C) and Hsp27 (B, D) phosphorylation levels following a 10-min stimulation of EA.hy926 cells with galectin-1, galectin-3 or both galectins (1 µg/ml each), by ELISA (A, B) and Western blots (C, D). For ELISAs, the data (mean +/− SEM) are shown as relative values compared with the control (no galectin addition), and significant differences are indicated (* p<0.05, ** p<0.01 and *** p<0.001). Quantification of Western blots was done using ImageJ (see Materials and Methods).

Figure 5. Modulation of VEGFR endocytosis by exogenous galectins in EA. hy926 cells.

The effects of exogenous galectins (1 µg/ml each) were evaluated by analysing the colocalisation between each receptor and EEA1 using the proximity ligation assay and an image analysis tool. Representative images of z-stacks of 7 fluorescent micrographs projected into a single phase-contrast image (original magnification: ×60) are shown. Signal/cell values are shown as relative values (mean +/− SEM) compared with the control (no galectin addition). The tables show the significance levels obtained by applying the standard Dunn procedure (post-hoc test) to compare all the pairs of experimental conditions, in order to avoid multiple comparison effects (NS  =  not significant: p>0.05). Scale bar: 20 µm.