Abstract 19: PlGF/VEGFR-1 dependent activation of the Dll4-Notch4/Ephrin B2 cascade contributes to liver vessel anomalies in hepatocellular carcinoma
Authors: Tijeras-Raballand Annemilai , Gramont Armand de , Hainaud Patricia , Contreres Jean-Olivier , Hénaff Carole Le , Pocard Marc , Dupuy Evelyne 
Published: May 2015

Introduction: Hepatocellular carcinoma (HCC), is characterized by severe vessel anomalies with an intense arterial blood supply (arterialisation), and acquisition ofa basal membrane rich in laminin by sinusoids(sinusoid capillarisation). A role of the VEGF-A/Dll4-Notch4/ephrin B2 cascade in tumor vessel anomalies has been demonstrated, raising the question of the nature of theVEGF receptor pathway implicated in triggering this cascade. This study was aimed at determining whether PlGF/VEGFR-1 or VEGF-A/VEGFR-2 pathways were implicated in the vessel anomalies observed during HCC development.

Material and Methods:In vitro, we used primary culture of HUVECs, isolated in our laboratory from human umbilical cords. HUVECs were cultures in EBM2 medium, complemented with 20% of FBS and 2ng/ml of FGF-2. To determine which pathway was predominant in the activation of the Dll4/Notch4 cascade, the effects of PlGF (a specific ligand of VEGFR-1), VEGF-E (a specific ligand of VEGFR-2) and VEGF-A (a ligand of both receptors) on the expression of the active form of Notch4 and Dll4 in HUVECs were compared by Western Blot. In vivo, () the differential expression of VEGF-A, PlGF and their receptors were evaluated by qRT-PCR and immunostainings in transgenic mice developing stage-defined HCC.

Results: In vitro, HUVECs stimulation by either VEGF-A, VEGF-E or PlGF increased Dll4 expression and Notch 4 activation. However, PlGF had the most important effect with a 5-fold increase of Dll4 expression level and a 4-fold increase in Notch 4 activation, compared to control HUVECs. Moreover, downregulation of VEGFR-1 by small interfering RNA, but not VEGFR-2, abrogated the effects of VEGF-A and PlGF stimulation on Dll4 expression and Notch4 activation. To confirm in vivothe prevalence of the PlGF/VEGFR-1 pathway in induction of vessel anomalies, we first evaluated the differential expression of PlGF, VEGF-A, VEGFR-1 and VEGFR-2 in our transgenic HCC model at different stages of the disease. We showed that VEGFR-1 increased with HCC progression and was expressed by macrophages and sinusoidal endothelial cells. Moreover, PlGF levels were maximal at the stage that coincides with the beginning of liver vessels anomalies in HCC. Silencing PlGF in vivo delayed tumor growth (p&lt;0.05), reduced arterial vessel length by 84% (p&lt;0.05), and sinusoids anomalies with a decrease in Dll4 and active Notch4 expression levels without affecting microvascular density.

Conclusions: We demonstrated that PlGF and VEGFR-1 played a key role in the development of vessel anomalies in HCC and that silencing PlGF prevents liver vessel anomalies and delays tumor growth. Our results bring a new insight in the mechanism responsible for vessel anomalies observed in HCC, giving a strong rationale for the development of new antiangiogenic strategies based on PlGF inhibition in HCC.

Citation Format: Annemilai Tijeras-Raballand, Armand de Gramont, Patricia Hainaud, Jean-Olivier Contreres, Carole Le Hénaff, Marc Pocard, Evelyne Dupuy. PlGF/VEGFR-1 dependent activation of the Dll4-Notch4/Ephrin B2 cascade contributes to liver vessel anomalies in hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 19. doi:10.1158/1538-7445.AM2014-19