Thierry Avramoglou

Antiproliferative capacity of synthetic dextrans on smooth muscle cell growth: the model of derivatized dextrans as heparin-like polymers

Proliferation of vascular smooth muscle cells (SMC) is postulated to be a key step in the pathogenesis of atherosclerosis or restenosis after vascular interventions such as angioplasty. Natural glycosaminoglycans, such as heparin and heparan sulfate, are known for their ability to inhibit SMC proliferation in vivo and in vitro. The antiproliferative activity of synthetic derivatized dextrans exhibiting heparin-like anticoagulant and anticomplement capacities have been investigated with rat aorta smooth muscle cells in culture. We report here that some derivatized dextrans grafted with benzylamide sulfonate moieties are potent antiproliferative agents for rat smooth muscle cell (SMC) in vitro. These synthetic polymers inhibit the SMC proliferation as well as heparin. The SMC growth inhibition is dose dependent, reversible and non-toxic. Highly anionic carboxylic dextrans are not capable of inhibiting the SMC growth, excluding a simple charge effect mechanism. Using fluorescent (DTAF) probes, we demonstrated that the synthetic antiproliferative polymers and heparin are internalized into the SMC. No binding or internalization was observed with native dextran devoid of antiproliferative capacity. We conclude that a suitable distribution of functional groups on the dextran backbone can simulate heparin activity in terms of antiproliferative capacity on SMC growth.

Inhibition by heparin and derivatized dextrans of Staphylococcus aureus adhesion to fibronectin-coated biomaterials

Recent data on cardiovascular device-centered infections suggest that some plasma and extracellular matrix proteins contribute to bacterial adhesion and colonization on biomaterials. We previously developed an in vitro assay to study the Staphylococcus aureus adhesion-promoting effect of surface-adsorbed fibronectin on flat PMMA coverslips coated with a monolayer amount of fibronectin. We screened the potential anti-adhesive properties of a group of substituted dextrans, previously shown to exhibit potent anticoagulant and anticomplementary activities. In comparison to unsubstituted dextran which showed no significant (< 20%) adhesion inhibition at 1 mg/ml, dextrans increasingly substituted with carboxylic and benzylamide groups (CMBD) exhibited increasing anti-adhesive activities. Three CMBD derivatives showing an increasing proportion (5-14%) of benzylamide groups showed inhibition of bacterial adhesion increasing from 33 to 51% at 1 mg/ml. Another category of substituted dextrans having a variable proportion (2-26%) of sulfonated benzylamide groups (CMBDS) produced active inhibition of S. aureus adhesion. In comparison to these heparin-like dextran derivatives, native heparin produced inhibition values of S. aureus adhesion which were intermediate between those of CMBD and CMBDS compounds. Furthermore, the anti-adhesive activity was still expressed when substituted dextrans were preincubated with fibronectin-coated PMMA but washed away at the time when radiolabeled bacteria were added to the adhesion assay. This indicates that the anti-adhesive effects of CMBDS could be exerted at the level of the S. aureus binding site of fibronectin. In conclusion, S. aureus adhesion on fibronectin-coated biomaterials can be efficiently blocked in vitro by soluble compounds such as dextran derivatives.

Derivatized dextran inhibition of smooth muscle cell proliferation.

Proliferation of vascular smooth muscle cells is postulated to be one of the key events in the pathogenesis of atherosclerosis or during the development of focal glomerular sclerosis. Several studies have suggested that the antiproliferative effects of heparin appear to be regulated by different structural determinants. Our experiments show that dextrans substituted with carboxylic and benzylamide sulphonate groups markedly inhibit the growth of smooth muscle cells in vitro. Studies on the structure-function relationships of these products to their effect on rat aorta smooth muscle cells are reported. The antiproliferative capacity is similar to that of heparin.

Synthesis and Characterization of a New Polysaccharide-graft-polymethacrylate Copolymer for Three-Dimensional Hybrid Hydrogels

Hybrid materials constituted by hydrophobic and hydrophilic biocompatible macromolecules are useful for biomedical applications. In this context, a well-known acrylic monomer (methyl methacrylate) was polymerized and grafted onto the polysaccharide dextran by the use of ceric ammonium nitrate as a redox initiator in aqueous nitric acid medium. The effects of concentrations of dextran, acrylic monomer, and ceric ions on the copolymerization yields were investigated in detail. The obtained polymers were studied by solubility measurements, Fourier transform infrared spectrometry, (13)C nuclear magnetic resonance spectroscopy, and viscosimetric analysis. Interestingly, we found conditions to form transparent and homogeneous thin films or 3D structures with hybrid properties. Indeed, the copolymer, but not dextran or PMMA, could be dissolved in water/THF (20/80 v/v). The thermomechanical properties of the resulting copolymer analyzed by differential scanning calorimetry and dynamic mechanical analysis showed the occurrence of a single glass-transition temperature and a marked difference with the two homopolymers. The cytocompatibility of the copolymer with human endothelial cells was evidenced by the normal cell adhesion, proliferation, and morphology after 5 days in culture on these gels. In conclusion, this type of copolymer with hybrid properties of two biocompatible macromolecules could be of great interest as a 3D scaffold or for coating in biomedical applications.

Effect of a derivatized dextran on human osteoblast growth and phenotype expression.

Water soluble derivatized dextran named E9 with a molecular weight of 45,000 g l-1 containing 58% methyl carboxylic acid unit, 19% benzylamide unit, and 26% sulfonate with a specific anticoagulant activity of 0.29 IU mg-1 was studied for its effects on human osteoblast growth and phenotype expression for short-term treatment. At concentrations between 1 ng ml-1 and 1 microgram ml-1 E9 has no effect on DNA synthesis whereas at higher concentrations DNA synthesis is inhibited in a dose related fashion (87% for 400 micrograms ml-1). For concentrations which do not modify osteoblast growth, E9 promotes alkaline phosphatase activity, type I collagen and osteocalcin synthesis with a maximum effect for 0.1-1 microgram ml-1. It has a synergistic effect with hPTH increasing AMPc. Moreover, osteonectin synthesis was enhanced in a dose-dependent manner between 0.1 and 5 micrograms ml-1. These results seem to indicate that E9 is able to stimulate human osteoblast phenotype expression and could be useful in clinical applications.

Low-molecular-weight dextran derivatives (f-CMDB) enter the nucleus and are better cell-growth inhibitors compared with parent CMDB polymers.

Carboxymethyldextrans-benzylamide (CMDB) are dextran derivatives that are statistically substituted with carboxymethyl and benzylamide groups. These molecules display a variety of biological effects, one of which is their inhibitory activity against mammary tumor cell growth, both in vitro and in vivo. We and others have previously shown that the effects of CMDB on cell growth are related to their ability to interact with the growth factor FGF-2. The binding modifies the conformation of FGF-2, leading to the suppression of its mitogenic activity. Here, the method previously reported to fragment natural polysaccharide fucans has been applied to CMDB (80,000 g/mol). f-CMDB (fragmented CMDB) of molecular weights from 6000 to 20,000 g/mol were found to be more potent inhibitors of MCF7 mammary tumor cell growth than high-molecular-weight CMDB. Confocal microscopy experiments using CMDB and f-CMDB labeled with the fluorophore DTAF (5-([4,6-dichlorotriazine-2-yl]amino) fluorescein) indicate that only low-molecular-weight f-CMDB penetrate into the nucleus of MCF7 cells. It is thus assumed that the better inhibitory properties demonstrated by f-CMDB, compared with CMDB, are related to their better ability to penetrate the nucleus and interact with nuclear targets, including topoisomerase II. The DNA relaxation properties of the latter are inhibited in vitro by both CMDB and f-CMDB. These findings could help us to develop models of low-molecular-weight oligosaccharide derivatives exhibiting better antiproliferative and antitumor properties.

Inhibitory effect of substituted dextrans on MCF7 human breast cancer cell growth in vitro.

Substituted dextrans can reproduce some of the properties of heparin and can thus be used to alter cellular growth. We studied the effect of heparin (H108), dextran (D), carboxymethylbenzylamide dextran (CMDB) and carboxymethylbenzylamide sulfonate dextran (CMDBS) on the growth of human mammary cells of the MCF7 tumor line. The cells were cultured in minimum Eagles medium containing 2% fetal calf serum without biopolymer, or with increasing concentrations of H108, D, CMDB or CMDBS. Growth curves were accurately based on cell counting using a Coulter counter. Cell distribution in the various phases of the cycle was analyzed by flow cytometry. Dose-dependent growth inhibitory effects (400–4000 μg/ml) were observed. The effect on MCF7 tumor cells was most apparent with CMDBS. The percentage of cells in the S phase decreased with preferential blocking In the G0/G1 phase. Pre-clinical studies can be anticipated as there is an absence of in vivo toxicity.

Films of dextran-graft-polybutylmethacrylate to enhance endothelialization of materials.

We have synthesized new structures obtained from amphiphilic copolymers of dextran and polybutylmethacrylate with the aim of endothelialization of biomaterials. Grafting of butylmethacrylate onto dextran has been carried out using ceric ammonium nitrate as initiator. Three copolymers were obtained (11, 30 and 37 wt.% dextran) and homogeneous thin films were successfully prepared. In contrast to dextran, the resulting films were stable in water, and copolymers characterized by Fourier transform infrared spectroscopy, differential scanning calorimetry and dynamic mechanical analysis showed evidence of hybrid properties between the parent homopolymers. Surfaces of films were smooth when analyzed by atomic force microscopy (roughness 2+/-1 nm) but greatly differed in their hydrophilicity by increasing the dextran content (water contact angle from 99 degrees to 57 degrees). In contrast to polybutylmethacrylate, where the proliferation of vascular smooth muscle cells (VSMCs) was excellent but that of endothelial cells was very low, the copolymer containing 11% of dextran was excellent for endothelial cells but very limited for VSMCs. An in vitro wound assay demonstrated that copolymer with 11% dextran is even more favorable for endothelial cell migration than tissue-culture polystyrene. Increasing the dextran content in the copolymers decreased the proliferation for both vascular cell types. Altogether, these results show that transparent and water-insoluble films made from copolymers of dextran and butylmethacrylate copolymers with an appropriate composition could enhance endothelial cell proliferation and migration. Therefore, a potential benefit of this approach is the availability of surfaces with tunable properties for the endothelialization of materials.

Inhibitory effect of functional dextrans on rat arterial smooth muscle cell growth in vitro

Abstract Derivatized dextrans substituted with carboxylic, benzylamide and sulfonate groups were tested in vitro for their antiproliferative activity on arterial rat smooth muscle cells (SMCs). Some of these sulfated polysaccharides, without potent anticoagulant activity, exhibit an inhibitory effect on cell growth as effective as heparin. A study of different molecular weight fractions, from 4000 to 180000g mol−1 obtained by gel filtration chromatography, shows that this inhibitory effect disappears below about 65 osidic units. The action of these sulfated polysaccharides is a time-dependent event which blocks the cell cycle at the G 0 G 1 phase with the largest effect exhibited during the first 4 h. From the kinetics of DNA synthesis experiments, it appears that the antiproliferative effect is caused by a delay in cells entering the S phase from the G 0 G 1 phase. Because these compounds have low anticoagulant activity and are non-cytotoxic, they may represent an inexpensive source of new types of agent to suppress intimai SMC hyperplasia.

Growth inhibition of human melanoma tumor cells by the combination of sodium phenylacetate (napa) and substituted dextrans and one Napa–dextran conjugate

We have studied the cytostatic effects of sodium phenylacetate (NaPA) in association with several substituted dextrans on human tumor melanoma 1205LU cells. We show that NaPA alone inhibits the growth of these cells (IC50 = 3.9 mM) while a weak inhibitory effect appears at a concentration of 37 μ M (10 μ g/ml) for a dextran methyl carboxylate benzylamide (LS17-DMCB). The precursors of LS17-DMCB [T40 Dextran and carboxymethyl dextran (LS17-DMC)] did not affect the growth of 1205LU cells. To potentiate the inhibitory activity of NaPA at low concentrations (below 5.6 mM), we have tested NaPA and LS17-DMCB in physical mixture (association) or linked together covalently (this conjugate is termed ‘LS17–NaPaC’). We have observed an increase of the 1205LU cell growth inhibition effect with NaPA in association (IC50 1.8 mM). For a concentration of 5 mM of NaPA (free in the case of association or linked in the case of conjugate), the association with dextran derivative exhibits a 4.6-fold higher efficacy than with NaPA alone (9 versus 41% surviving fraction), while the conjugate is 1.3-fold smaller (52% growth inhibition). By performing isobologram analysis of the IC50 data, we have shown a synergistic effect for a particular molar ratio of NaPA and LS17-DMCB (NaPA:LS17-DMCB = 0.35).