Elsevier

Journal of Hepatology

Volume 58, Issue 2, February 2013, Pages 319-328
Journal of Hepatology

Research Article
The placental growth factor as a target against hepatocellular carcinoma in a diethylnitrosamine-induced mouse model

https://doi.org/10.1016/j.jhep.2012.09.032Get rights and content

Background & Aims

The placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family known to stimulate endothelial cell growth, migration and survival, attract angiocompetent macrophages, and determine the metastatic niche. Unlike VEGF, genetic studies have shown that PlGF is specifically involved in pathologic angiogenesis, thus its inhibition would not affect healthy blood vessels, providing an attractive drug candidate with a good safety profile.

Methods

We assess whether inhibition of PlGF could be used as a potential therapy against hepatocellular carcinoma (HCC), by using PlGF knockout mice and monoclonal anti-PlGF antibodies in a mouse model for HCC. In addition, the effect of PlGF antibodies is compared to that of sorafenib, as well as the combination of both therapies.

Results

We have found that both in a transgenic knockout model and in a treatment model, targeting PlGF significantly decreases tumor burden. This was achieved not only by inhibiting neovascularisation, but also by decreasing hepatic macrophage recruitment and by normalising the remaining blood vessels, thereby decreasing hypoxia and reducing the prometastatic potential of HCC.

Conclusions

Considering the favourable safety profile and its pleiotropic effect on vascularisation, metastasis and inflammation, PlGF inhibition could become a valuable therapeutic strategy against HCC.

Introduction

Every year approximately 500,000 patients are diagnosed with hepatocellular carcinoma (HCC), making it the 5th most common and 3rd most deadly cancer worldwide. Currently, no curative option exists for advanced HCC and systemic treatment is limited due to the high multidrug resistance [1].

Significant progress on the treatment of HCC has been made by the introduction of sorafenib [2]. Sorafenib is a small molecular inhibitor targeting tyrosine protein kinases, causing both an antiproliferative and antiangiogenic effect [3]. It is currently used as the standard-of-care for advanced HCC patients, yet it often causes severe adverse effects including diarrhoea, weight loss, hand–foot skin reaction, and fatigue [4], [5].

Another weakness of current antiangiogenic therapies is that tumors tend to escape their treatment by upregulating alternative angiogenic pathways. As a result, several cases have been reported of resistance against sorafenib [6], [7], sunitinib [8], [9], and vascular endothelial growth factor (VEGF) inhibitors [10], [11], [12], [13], [14]. Furthermore, hypoxic conditions created by antiangiogenic therapy may select for more invasive tumor variants, better adapted to survive, and proliferate [15], [16], [17]. Hypoxia is linked to metastasis, regulating several steps of the metastatic process, such as invasion through the basement membrane and extracellular matrix, increasing production of fibronectin and regulation of genes that promote the pre-metastatic niche [18]. Thus, there is a vast need of new angiogenic targets that could prevent these negative effects.

The placental growth factor (PlGF) is a member of the VEGF family, known to stimulate endothelial cell growth, migration, and survival [19], [20], [21]. PlGF is expressed in several cell types, including endothelial cells, hepatic stellate cells, and activated macrophages [22], [23], [24]. It attracts angiocompetent macrophages and bone marrow progenitor cells and determines the metastatic niche [19], [20]. Unlike VEGF, PlGF binds to VEGF-receptor-1 (VEGFR1), and its co-receptors neuropilin-1 and 2 [25], [26]. Besides its effect on tumor vascularisation, PlGF can alter tumor-associated macrophage (TAM) polarisation [27]. Tumors tend to attract M2 macrophages, which produce angiogenic and proliferative factors, enhancing tumor progression, while M1 macrophages are associated with an antitumoral response [28].

Genetic studies have shown that PlGF is specifically involved in pathologic angiogenesis [19], [21]. Therefore, its inhibition would not affect healthy blood vessels, providing an attractive drug candidate with a good safety profile [29]. In this study, we assess whether inhibition of PlGF could serve as a therapeutic agent in a diethylnitrosamine (DEN)-induced mouse model for HCC, in which well-vascularised tumors occur after 25 wk and a time-dependent increase of PlGF is observed [30]. In our study, we focus on the effect of PlGF inhibition on angiogenesis, metastasis, inflammation, and vascular morphology.

Section snippets

HCC induction

Mice received intraperitoneal injections of DEN (Sigma–Aldrich, Bornem, Belgium) once a week or saline as previously described [31]. This model induces HCC after 25 wk.

PlGF inhibition

An anti-PlGF monoclonal antibody (clone 5D11D4; referred to as αPlGF) specifically recognising mouse PlGF-2 was obtained from Thrombogenics, Belgium. Mice that received DEN for 25 wk developed HCC and were subsequently treated with 25 mg/kg αPlGF diluted in saline (2×/week, intraperitoneal, n = 12), 10 mg/kg sorafenib diluted in saline

PlGF knockout study

Silencing of Pgf significantly improved survival (p <0.01) (Fig. 1A). Macroscopically, PlGFKO-mice developed less tumors compared to their WT counterparts, respectively 0.11 ± 0.11 in 20 wk PlGFKO vs. 2.25 ± 0.59 in 20 wk WT; 0.63 ± 0.42 in 25 wk PIGFKO vs. 4.29 in 25 wk WT; and 7.75 ± 1.03 in 30 wk PlGFKO vs. 15.40 ± 1.04 in 30 wk DEN WT (p <0.05). This was reflected in a decreased microscopic number of tumors and dysplastic lesions (Table 1). Small cell dysplasia was found throughout the liver, starting from 20

Discussion

Antiangiogenic treatment has opened a new era in the field of anticancer therapy. While most studies have focussed on inhibiting VEGF and its receptors, we have chosen to investigate the role of the VEGF homologue PlGF, known for its sole involvement in pathologic angiogenesis, in the pathogenesis of HCC. HCC is a solid tumor greatly depending on neovascularisation to support its growth. Therefore, antiangiogenic treatment with sorafenib has shown substantial improvement in patients with

Financial support

F. Heindryckx received a scholarship (FWO09/ASP/161) from the Fund for Scientific Research (FWOFlanders). S. Coulon received a scholarship (BOF 09/24J/012) from the University Ghent Research Fund (BOF). H. Van Vlierberghe and I. Colle received a fundamentally clinical mandate of the Fund for Scientific Research (FWOFlanders). P. Carmeliet received Long term Structural funding Methusalem – by the Flemish Government, Interuniversity Attraction Poles Program – Federal Government – P06/30, Fund for

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgements

The authors would like to thank J. Dupont and E. Bogaerts for their excellent assistance during the experiments. Microarrays were performed by the VIB nucleomics core.

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