Conclusions
TNF-α promoted PlGF synthesis in BeWo cells and regulated angiogenesis via synergy of the PlGF/VEGFR1 and VEGF-A/VEGFR2 axes.
Methods
After incubation with TNF-α (10-105 pg/mL), the expression of PlGF and VEGF-A in BeWo cells were assessed by ELISA and RT-PCR. HUVEC tube formation assays were conducted to assess the angiogenic activity of the conditioned medium. The phosphorylation status of VEGFR1 and VEGFR2 in HUVECs under the stimulation of the conditioned medium was assessed by immunoprecipitation and immunoblotting. The same experiments were repeated with recombinant PlGF and VEGF-A to confirm the effects of the growth factors.
Results
Low levels (10-102 pg/mL) of TNF-α enhanced the mRNA and protein levels of PlGF, but the changes in VEGF-A levels were not significant. HUVEC tube formation was promoted by the conditioned medium, and those effects were inhibited by the anti-VEGFR1 antibody and PlGF-siRNA. VEGFR2 was significantly phosphorylated by the conditioned medium, while the effect on VEGFR1 phosphorylation was very weak. HUVEC tube formation was incomplete when recombinant PlGF was used; however, the addition of PlGF promoted the effects of VEGF-A. The addition of PlGF along with VEGF-A also stimulated VEGFR2 phosphorylation. Conclusions: TNF-α promoted PlGF synthesis in BeWo cells and regulated angiogenesis via synergy of the PlGF/VEGFR1 and VEGF-A/VEGFR2 axes.