Effect of fucoidan on fibroblast growth factor-2-induced angiogenesis in vitro

Abstract

Fucoidans are sulfated polysaccharides extracted from brown marine algae. A purified fucoidan fraction exhibits the same venous antithrombotic activity as heparin in rabbits, but with a lower anticoagulant effect. Because of its heparin-like structure, we postulated that fucoidan might modulate heparin-binding angiogenic growth factor activity. We thus studied its effect, at antithrombotic concentrations, on fibroblast growth factor (FGF)-2-induced proliferation and differentiation of human umbilical vein endothelial cells. The fucoidan effect on endothelial cell differentiation was evaluated by studying the expression of surface proteins (i.e. integrin, adhesion molecule) known to be modulated by FGF-2 and involved in angiogenesis, and by quantifying closed areas delimited by vascular tubes formed on reconstituted basement membrane. Fucoidan had no modulatory effect on the mitogenic activity of FGF-2, but significantly increased tubular structure density induced by FGF-2. Fucoidan alone increased α₆ integrin subunit expression with only partially organized tubular structure. In the presence of FGF-2, fucoidan enhanced α₆, β₁ and PECAM-1 and inhibited α_vβ₃ integrin expression. Heparin had no effect in these systems. The most striking effect of fucoidan was observed on α₆ expression and tube formation was abolished by monoclonal anti-α₆ antibodies. Fucoidan plus FGF-2 effect on α₆ expression was markedly decreased by monoclonal anti-FGF-2 antibodies, indicating that fucoidan acts mainly via FGF-2. These results show that, at antithrombotic concentrations, contrary to heparin, fucoidan can enhance vascular tube formation induced by FGF-2 with a modulation of the expression of surface proteins (mainly α₆) involved in angiogenesis.

Introduction

Fucoidans are sulfated polysaccharides extracted from marine brown seaweed. We have previously shown that they exhibit the same antithrombotic activity as heparin in rabbits (evaluated in Wessler model), with lower anticoagulant activity and a lesser hemorrhagic risk [1]. These experiments were performed with standard heparin and a fucoidan fraction of the same molecular weight [1]. Given its polyanionic characteristics comparable to those of heparin, fucoidan can interact with a large number of basic amino acid-rich proteins such as blood coagulation inhibitors (antithrombin, heparin cofactor 2, etc.) [2], explaining its anticoagulant activity, and with heparin-binding growth factors (fibroblast growth factor-1, fibroblast growth factor-2, etc.) involved in cell metabolism [3]. Fibroblast growth factor-2 (FGF-2), which is ubiquitous, is thought to play a major angiogenic role in vascular wound repair and collateral vessel formation [4]. In vivo, FGF-2 is stored bound to low-affinity sites provided by heparan sulfate proteoglycans, namely perlecan, located in the extracellular matrix, and syndecan, located on the cell surface [5]. Yayon et al. [6] have shown that heparan sulfate proteoglycans are low-affinity coreceptors for FGF-2 binding to their high-affinity receptors, and constitute a trimolecular complex which is more stable than the complex between FGF-2 and the receptor alone. FGF-2 is released from heparan sulfate chains by heparanase or by competitive binding of soluble heparin-like molecules [5], [7]. At the cellular level, the FGF-2 release pathway is unclear, as FGF-2 has no signal peptide. It has been suggested that intracellular FGF-2 may be released passively in response to mild cell damage [8] and some stress conditions associated with tissue injury [9]. In these conditions, released FGF-2 can stimulate endothelial cells. FGF-2 induces many endothelial cell modifications involved in angiogenesis (for a review, see Ref. [10]), namely: (i) increased production of proteases (u-PA, t-PA) and their receptors (u-PAR) involved in basement membrane degradation, (ii) stimulation of proliferation and migration (involving  α_vβ₃) during vessel elongation and (iii) cell differentiation to form tubes expressing or overexpressing cell surface proteins (integrins and adhesion molecules). Angiogenesis also depends on interactions between surface proteins and extracellular matrix proteins. In vitro models of angiogenesis using Matrigel (a reconstituted basement membrane rich in laminin) or collagen gel have allowed the identification of surface proteins involved in tube formation, such as α₆ and β₁ integrin subunits [11] and the PECAM-1 adhesion molecule [12]. Many heparan sulfate proteoglycans such as perlecan or syndecan have a potentiating effect on FGF-2 angiogenic activity [13], [14]. Heparin has a more controversial effect on angiogenesis, depending on the experimental conditions: it can stimulate collateral circulation formation in ischemic conditions in the presence of FGF-2 [4] and inhibit angiogenesis when administered in combination with steroid compounds [15]. Fucoidan, like heparin, is also a potent antiproliferative agent for rat arterial smooth muscle cells [16], and this could be of interest for the prevention of restenosis. Compounds with antithrombotic activity, weak anticoagulant effects and inhibitory effects on smooth muscle cell proliferation are candidate drugs against restenosis, especially if they are able to promote angiogenesis and to restore the endothelial monolayer. We thus studied the effect of fucoidan on endothelial cell proliferation, surface protein expression and differentiation into vascular tubes in the presence of FGF-2.