A placenta growth factor 2 variant acts as dominant negative of vascular endothelial growth factor A by heterodimerization mechanism

Abstract

Angiogenesis is one of the crucial events for cancer development and growth and vascular endothelial growth factor (VEGF) family plays an essential role in this biological phenomenon. The members of VEGF family mainly involved in angiogenesis are VEGF-A, VEGF-B and placental growth factor (PlGF), which exert their activity through the binding and activation of two VEGF receptors, VEGFR-1 and VEGFR-2. Human VEGF-A and PlGF are expressed in different isoforms and have the peculiarity to form heterodimer if co-expressed in the same cell. The difference of two main human PlGF isoforms, PlGF1 and PlGF2, consist in the exclusive ability of PlGF2 to bind heparin and Neuropilin receptors. As previously reported for PlGF1 isoform, we have generated a PlGF2 variant named PlGF2 -DE, in which the residues D(72) and E(73) were substituted with alanine, that is unable to bind and activate VEGFR-1 but is still able to heterodimerize with VEGF. Here we report that overexpression in VEGF-A producing human tumor cell line derived from ovarian carcinoma (A2780) of PlGF2-DE variant by stable transfection, significantly reduces the production of VEGF-A homodimer via heterodimerization, determining a strong inhibition of xenograft tumor growth and associated neoangiogenesis, as well as significant reduction of monocyte-macrophage infiltration. Conversely, the overexpression of PlGF2wt, also reducing the VEGF-A homodimer production comparably to PlGF2-DE variant through the generation of VEGF-A/PlGF2 heterodimer, does not inhibit tumor growth and vessel density compared to control, but induces increase of monocyte-macrophage infiltration. Interestingly the comparison of PlGF2wt with PlGF1wt overexpression evidences a significant reduction of monocyte-macrophages recruitment as unique difference among the activity of the two PlGFwt isoforms. Therefore, the 'less soluble' PlGF2 shows a limited potential in monocyte-macrophages recruitment. In conclusion data here reported demonstrate that PlGF-DE variant acts as 'dominant negative' of VEGF-A independently from the PlGF isoform utilized, that the expression of active PlGF2 homodimer and VEGF-A/PlGF2 heterodimer is sufficient to rescue pro-angiogenic activity lost for reduction of VEGF-A due to heterodimerization mechanism, and that PlGF2 shows lower activity into recruitment of monocyte-macrophage cells compared to PlGF1 isoform.

Figure 1

In vitro proliferation of A2780 stable clones and binding properties of PlGF2-DE. A. A2780 stable clones were seeded in 96-well plates (2000 cells/ cm²) and cell proliferation was evaluated using the CellTiter Aqueous One Cell Proliferation Assay (Promega) at indicated time. B. Binding of VEGF-A homodimer (2.5 to 10 ng/ml), VEGF-A/PlGF2-DE heterodimer (7.5 to 30 ng/ml) and PlGF2-DE homodimer (81.25 to 325 ng/ml) present in the supernatant of A2780-PlGF2-DE stable clone, to coated Flt-1 (0.5 μg/ml) in ELISA based assay. For both experiments each point was carried out in triplicate and the data are represented as the mean of two experiments ± SEM. (C) Western blot analysis of Flt-1 phosphorylation (anti-P-Flt-1) induced by 20 ng/ ml of PlGFwt isoforms or 100 ng/ml of PlGF-DE isoforms, on starved 293-Flt-1 cells. One hundred μg of the cell protein extracts were analyzed. Normalization was performed with ant-Flt-1 antibody.

Figure 2

PlGF2-DE variant inhibits tumor growth and neoangiogenesis. A. Exponentially growing A2780 stable clones were subcutaneously injected into 8-week-old CD1 nude athymic mice (3×10⁶ cells, n=8 per groups). Tumor volume (TV) was measured two times a week. The volume of tumors overexpressing the DE variant of both PlGF isoforms was strongly and significantly reduced at day 21 (*,§ p<0.0001 vs A2780-pCDNA3, A2780-PlGF2wt and A2780-PlGF1wt). Data are represented as the mean ± SEM. B. Vessel density was calculated analyzing five optical fields for each tumor, counting CD31 positive vessels. *,§p<0.0001 vs A2780-pCDNA3; ¶p<0.001 vs A2780-PlGF1wt and A2780-PlGF2wt; #p<0.001 vs A2780-PlGF1wt; ^p<0.005 vs A2780-PlGF2wt. Data are represented as the mean ± SEM. C. Representative pictures of CD31 staining of A2780 tumors. Scale bars represent 50 μm.

Figure 3

Monocyte-macrophage infiltration in tumors overexpressing PlGF2-DE or PlGF2wt. The area of monocyte-macrophage infiltration in the xenograft tumors was evaluated by immunostaining with anti-F4/80 antibody analyzing five optical fields for each tumor and data are represented as the mean ± SEM. A. A2780-PlGF2-DE tumors showed a reduced F4/80 positive area compared to A2780-pCDNA3 tumors (*p=0.0060) and similar to that observed in A2780-PlGF1-DE tumors (§p=0.0025 vs A2780-pCDNA3). Conversely, both A2780-PlGF2wt and A2780-PlGF1wt tumors showed an increase of F4/80 positive cells area if compared to A2780-pCDNA3 (#p=0.0068 and ¶p<0.0001 respectively). The F4/80 positive area in A2780-PlGF2wt tumors resulted significantly reduced if compared to A2780-PlGF1wt tumors (^p=0.0424). B. Representative pictures of F4/80 staining of A2780 tumors. Scale bars represent 50 μm.