Tumor necrosis factor-α regulates angiogenesis of BeWo cells via synergy of PlGF/VEGFR1 and VEGF-A/VEGFR2 axes

Highlights

• Low TNF-α stimulated PlGF synthesis in BeWo, not affecting VEGF-A.

• High TNF-α inhibited both PlGF and VEGF-A synthesis in BeWo.

• PlGF/VEGFR1 did not independently promote endothelial angiogenesis.

• PlGF/VEGFR1 enhanced VEGF-A/VEGFR2 promoting effects of endothelial angiogenesis.

• TNF-induced PlGF synthesis was associated with NF-kB signaling pathway.

Abstract

Objectives

Tumor necrosis factor-alpha (TNF- α) promotes tumor growth by enhancing tumor angiogenesis; however, the effects on choriocarcinoma remain unknown. We investigated the effects of TNF-α on the production of placental growth factor (PlGF) and vascular endothelial growth factor-A (VEGF-A) in BeWo cells and also examined its significance on the interactions with the endothelial cells by using human umbilical vein endothelial cells (HUVECs).

Materials & 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.

Introduction

Tumor microenvironment plays an important role in tumor progression. It has been accepted that a close interaction of cancer cells with the inflammatory microenvironment contributes to tumor growth and survival [1,2]. Tumor necrosis factor-alpha (TNF-α) is a potent pro-inflammatory cytokine and was originally reported as a cytotoxic molecule that induces apoptosis [3,4]. A high-dose of TNF-α confers anti-tumor activity and has been used for treatment of solid tumors [5]. By contrast, there is growing evidence to suggest that a low-dose of TNF-α acts as a tumor promoter [6,7]. Shaarawy et al. reported that patients with progressive choriocarcinoma had a significant elevation of serum TNF-α [8]. However, the underlying mechanism of the tumor promoting activity induced by TNF-α in choriocarcinomas has remained unknown. Various mechanisms have been suggested by which TNF-α promotes tumor growth, including enhancing autocrine growth, invasion, and angiogenesis [6]. Angiogenesis is an essential process for the spread of tumors and the increased expressions of proangiogenic factors are associated with tumor growth. TNF-α is able to promote tumor angiogenesis by inducing various proangiogenic factors [6,9,10].

BeWo cells were originally derived from human choriocarcinoma and established as a cell line to study endocrine function in vitro [11]. There has been little evidence on the effects of TNF-α on BeWo cells. We have previously reported that a low-dose of TNF-α stimulated proliferation and promoted survival of BeWo cells, partially in combination with insulin-like growth factor-I [12]. In the present study, we investigated the effects of TNF-α on the angiogenesis of choriocarcinomas using BeWo cells.

Vascular endothelial growth factor-A (VEGF-A) and placental growth factor (PlGF) are both members of the VEGF family and are known to be produced by choriocarcinomas [13]. PlGF binds to VEGF receptor 1 (VEGFR1/Flt-1) selectively, while VEGF-A can bind both to VEGFR1 and VEGF receptor 2 (VEGFR2/Flk-1) [14,15]. Both PlGF and VEGF-A have been proposed to play an important role in the regulation of endothelial angiogenesis [16].

In this study, we investigated the effects of TNF-α on the production of PlGF and VEGF-A in BeWo cells. Furthermore, we also sought to elucidate the angiogenetic roles of PlGF and VEGF-A in the microenvironment of BeWo cells and the endothelial cells.