Fucans, sulfated polysaccharides extracted from brown seaweeds, inhibit vascular smooth muscle cell proliferation. I. Comparison with heparin for antiproliferative activity, binding and internalization.

Smooth muscle cell (SMC) proliferation is inhibited both in vivo and in vitro by heparin. However, the precise mechanisms of action are still not understood. The analogy between two sulfated polysaccharides, heparin and fucan, has led us to compare in detail their effects on SMC growth. We have prepared and characterized a 19 kDa fucan fraction from brown seaweed, Ascophyllum nodosum. Fucan affects the growth of SMCs in a time- and dose-dependent, reversible and non-toxic fashion. As determined by cell counting, [3H]thymidine incorporation, and microcytofluorimetry analysis, heparin was less active than fucan in inhibiting SMC growth. Fucan and heparin act by preferential blocking of G0/G1, thus decreasing the G0/S transition. Binding experiments with [125I]fucan indicated saturable, unlabeled-fucan displaceable binding sites with an apparent Kd of 30 nM. Moreover, displacement experiments performed with various polysaccharides revealed that antiproliferative compounds interacted with these membrane sites, but non-antiproliferative polysaccharides (dextran, chondroitin sulfate) did not, providing evidence of a correlation between binding to SMCs and their antiproliferative activity. When cells were exposed at 37 degrees C to a fluorescent 5-([4,6-dichlorotriazin-2-yl]-amino)fluorescein (DTAF)-fucan, internalization occurred and punctate vesicles were observed which accumulated rapidly in the perinuclear region as previously reported for heparin. Nuclear preparations (membranes + contents) of cultured SMCs previously incubated with radiolabeled heparin or fucan indicated the presence of radioactivity, suggesting an antiproliferative action of both polysaccharides at the nuclear level. Collectively, these observations indicated that fucan and heparin share some similar mechanisms of action, such as SMC growth inhibition, binding, and internalization. In the accompanying paper (Logeart et al., Eur. J. Cell Biol. 74, 1997, this issue), we describe the effect of fucans of different molecular weights and conclude that there is no direct link between polysaccharide degradation and the antiproliferative effect on SMCs.

Fucans, sulfated polysaccharides extracted from brown seaweeds, inhibit vascular smooth muscle cell proliferation. II. Degradation and molecular weight effect.

Fucan, a sulfated polysaccharide extracted from brown seaweeds, inhibits smooth muscle cell (SMC) proliferation with a higher antiproliferative activity than heparin (Logeart et al., Eur. J. Cell Biol. 74, 1997, this issue). In order to investigate the structure-activity relationship of fucan on SMC growth, we have prepared by size exclusion chromatography fucan fractions of various molecular masses ranging from 5.5 to 556 kDa. Our experiments showed that the antiproliferative activity is dependent on the molecular weight of the polysaccharide. The molecular weight threshold indicated that about 30 saccharidic units on fucan were necessary to give the antiproliferative activity on SMCs. A kinetics study of DNA synthesis using tritiated thymidine uptake was also performed with different molecular weight fucan fractions. Although all tested fractions acted as soon as the cells enter the first cell cycle, the duration and potency of action varied. Moreover, displacement experiments of iodinated fucan revealed that the low molecular fucan fraction interacted weakly with the binding sites. Finally, gel permeation chromatography of internalized radiolabeled heparin and fucans was performed with SMCs. A rapid degradation of internalized heparin was observed, whereas only low molecular weight fucan fractions were partially degraded by SMCs. Together, these results indicate the significance of molecular weight on the antiproliferative activity of fucans on SMCs, and might help to understand their mechanism of action. In addition, the degradation experiments with internalized heparin and fucans ruled out a direct link between polysaccharide degradation and the antiproliferative effect on SMCs.

Interactions of functionalized dextran-coated liposomes with vascular smooth muscle cells.

Synthetic polymers are commonly used in the medical field as implants, polymeric drugs, or drug delivery systems. Among them, bioactive sulfated polysaccharides such as chemically modified dextrans are described to exhibit various properties including the inhibition of smooth muscle cell (SMC) growth. SMCs are key cellular components involved in the physiopathology of the vascular walls especially in atherosclerosis or after vascular surgeries. Interestingly, binding sites on vascular SMCs were already observed for an antiproliferative functionalized dextran (FDx). In this context, we hypothesized that this bioactive polymer could be used as a targeting moiety on the surface of drug delivery systems. In this work, liposomes constituted of phosphatidylcholine, phosphatidylethanolamine and cholesterol (70/10/20 mol.%) were prepared and coated with FDx hydrophobized by a cholesterol anchor (CholFDx) which penetrates the lipid bilayer during the liposome formation. The liposome interactions with SMCs were then followed using radiolabeled liposomes and fluorolabeled liposomes. Results of radioactivity on SMCs indicated higher interactions with CholFDx-coated liposomes as compared to uncoated liposomes. The fluorescence of cells incubated with fluorolabeled CholFDx-coated liposomes also evidenced the liposome binding on SMC membranes. These data demonstrated that liposomes coated with FDx interacted with vascular SMCs. Consequently, the coating with such bioactive polymers appears promising for the design of new drug delivery systems for the targeting of vascular cells.

Synthesis and characterization of highly sensitive heparin probes for detection of heparin-binding proteins.

Three labeled heparin species were synthesized as probes for heparin-binding protein detection. Heparin conjugated with 5([4,6-dichlorotriazin-2-yl]amino)fluorescein can be iodinated to a high specific activity. This probe specifically detected 40 pg histone on a dot blot without affinity purification. Heparin biotinylated on its naturally occurring primary amino groups also detected known heparin-binding proteins in a specific manner. This probe detected lower amounts of collagen I and basic fibroblast growth factor on nitrocellulose membranes than did the iodinated probe, with comparable detection times. To create more attachment sites for biotin, we covalently attached amino groups to the hydroxyl groups of heparin using 3-bromopropylamine hydrobromide. After biotinylation, the amino-rich probe detected heparin-binding proteins at the same or higher sensitivity as the biotinylated native heparin probe, using 100-fold less probe and much shorter detection times. This method of labeling is generally applicable to other polysaccharides, and would be useful when the amount of ligand is limited. We show that these three probes detect essentially the same spectrum of proteins in detergent extract of smooth muscle cell plasma membrane, and expect them to be useful probes for detection of cell-surface heparin receptors.

Fluorescent and radiolabeling of polysaccharides: binding and internalization experiments on vascular cells.

Glycosaminoglycans (GAGs) such as heparan sulfates are complex carbohydrate polymers. These structural components of the extracellular matrix are essential for the adhesion, migration, and regulation of cellular growth. To understand the physiological role of GAGs and GAG analogues, a practical approach consists of labeling and detecting them in cell extracts, or analyzing binding domains and their distributions into the cells. We propose a convenient and reliable method for preparing and labeling amino-enriched, polysaccharides with the fluorescent derivative 5-[(4,6-dichlorotriazine-2-yl)amino]-fluorescein (DTAF). Radioiodination is then performed on the DTAF moiety. This method was applied to polysaccharides known to inhibit vascular smooth-muscle cell (SMC) proliferation such as functionalized dextrans derived from poly(alpha 1-6 glucose) and fucan, poly(L-fucose 4-sulfate) extracted from brown seaweed. Using autoradiography and confocal microscopy, we observed the fixation and internalization of labeled antiproliferative products in SMCs from rat aorta. These probes can be useful for the understanding of polysaccharide-cell interactions. In addition, the method presented here can be applied to various synthetic or natural biomedical materials.