Assessment of Heparanase-Mediated Angiogenesis Using Microvascular Endothelial Cells: Identification of λ-Carrageenan Derivative as a Potent Anti Angiogenic Agent.

Nicolas Poupard,Hugo Groult,Pamela Badarou,Nicolas Bridiau,Catherine Grillon,Frédéric Sannier,Thierry Maugard,Ingrid Fruitier-Arnaudin,Claudine Kieda,Stéphanie Bordenave-Juchereau,Jean-Marie Piot,Fabienne Fasani

doi:10.3390/md15050134

Abstract

Heparanase is overexpressed by tumor cells and degrades the extracellular matrix proteoglycans through cleavage of heparan sulfates (HS), allowing pro-angiogenic factor release and thus playing a key role in tumor angiogenesis and metastasis. Here we propose new HS analogs as potent heparanase inhibitors: Heparin as a positive control, Dextran Sulfate, λ-Carrageenan, and modified forms of them obtained by depolymerization associated to glycol splitting (RD-GS). After heparanase activity assessment, 11 kDa RD-GS-λ-Carrageenan emerged as the most effective heparanase inhibitor with an IC50 of 7.32 ng/mL compared to 10.7 ng/mL for the 16 kDa unfractionated heparin. The fractionated polysaccharides were then tested in a heparanase-rich medium-based in vitro model, mimicking tumor microenvironment, to determine their effect on microvascular endothelial cells (HSkMEC) angiogenesis. As a preliminary study, we identified that under hypoxic and nutrient poor conditions, MCF-7 cancer cells released much more mature heparanase in their supernatant than in normal conditions. Then a MatrigelTM assay using HSkMEC cultured under hypoxic conditions in the presence (or not) of this heparanase-rich supernatant was realized. Adding heparanase-rich media strongly enhanced angiogenic network formation with a production of twice more pseudo-vessels than with the control. When sulfated polysaccharides were tested in this angiogenesis assay, RD-GS-λ-Carrageenan was identified as a promising anti-angiogenic agent.

Highlights

It is well known that solid tumor growth beyond 1–2 mm3 is dependent on angiogenesis, the sprouting of new blood vessels from preexisting ones to perfuse the tumor [1]
We investigated onthe oneeffect hand, of thethe effect sulfated onpolysaccharides the angiogenesis development during the firstduring seven the hours, of polysaccharides the low molecular weights (LMW) sulfated on the angiogenesis development firstwhen seven the cellularhours, activity is the on the hand, and, the number of pseudo formed t = 9.5 h, when thehighest cellularand, activity is other the highest on the other hand,vessels the number of at pseudo vessels formedreached at t = 9.5a h, when angiogenesis a plateau
We investigated the heparanase inhibition potential of sulfated polysaccharides submitted to depolymerization and glycol splitting

Summary

Introduction

It is well known that solid tumor growth beyond 1–2 mm is dependent on angiogenesis, the sprouting of new blood vessels from preexisting ones to perfuse the tumor [1]. Mar. Drugs 2017, 15, 134; doi:10.3390/md15050134 www.mdpi.com/journal/marinedrugs. Mar. Drugs 2017, 15, 134 promotes the tumor access to the nutrients and oxygen required for its development, opening in the meantime, the way to metastasis. It has been shown that hypoxia, the condition where cells suffer from oxygen deprivation, stimulates the expression of these factors [2,3]

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