gms | German Medical Science

Objective: VEGF-R3 plays an important role in developmental and lymphatic angiogenesis and is expressed in high-grade gliomas, but not in the normal brain. Herein, we investigated the role of VEGF-R3 as a potential specific and direct marker for tumor associated blood-vessels and explored if VEGF-R3 expression is maintained after anti-angiogenic therapy with the VEGF-A blocking antibody bevacizumab. Furthermore, we studied the anti-proliferative, anti-migratory and anti-angiogenic effects of the small-molecule MAZ51 which is a VEGF-R3 inhibitor in glioma cells and glioblastoma-derived endothelial cells (gbECs).

Method: PCR was performed for VEGF-R2, -R3 and their ligands VEGF-A, -C and -D in different malignant glioma cell lines (U87, U251, U373) as well as gbECs and endothelial cells from non-neoplastic tissue (HUVEC). All cell lines used have been stained immunocytochemically and analyzed by FACS for VEGF-R3 expression. Specimens of normal human brains as well as GBMs pre- and post-bevacizumab treatment were assessed for VEGF-R3 expression using immunohistochemistry. Glioma cells, gbECs and HUVEC were treated with bevacizumab, VEGF-R2 and -R3 blocking antibodies or with MAZ51. Cell numbers were quantified with the sulforhodamine-B (SRB)-assay and migration-rates were assessed with a wound healing assay. Angiogenesis of gbECs and HUVEC was evaluated in vitro using a matrigel-based vessel formation assay.

Results: Expression of VEGF-R3 was found in all tested cell lines on mRNA and protein level. Immunohistochemical evaluation showed that VEGF-R3 is absent from healthy brain, but is expressed on intratumoral blood vessels and on tumor cells in GBM. VEGF-R3 expression is still maintained after anti-angiogenic treatment with bevacizumab. Tumor cell proliferation and migration were significantly inhibited by MAZ51, whereas extracellular VEGF-A, -R2 and -R3 inhibition by specific antibodies had no impact. MAZ51 significantly reduced the ability of gbECs to form tube-like structures, while blocking the extracellular VEGF-R2/-R3 system showed no effect. In contrast control cells (HUVECs) showed the opposite effect.

Conclusions: The small molecule MAZ51 is a potent blocker of VEGF-R3 phosphorylation thereby reducing tumor cell proliferation and migration as well as exerting anti-angiogenic effects selectively in glioma-associated endothelial cells. Therefore, inhibition of VEGF-R3 may be a promising therapeutic approach in glioma cells and associated blood-vessels.