A Screening Approach to Assess the Impact of Various Commercial Sources of Crude Marine λ-Carrageenan on the Production of Oligosaccharides with Anti-heparanase and Anti-migratory Activities.

Oligosaccharides derived from λ-carrageenan (λ-COs) are gaining interest in the cancer field. They have been recently reported to regulate heparanase (HPSE) activity, a protumor enzyme involved in cancer cell migration and invasion, making them very promising molecules for new therapeutic applications. However, one of the specific features of commercial λ-carrageenan (λ-CAR) is that they are heterogeneous mixtures of different CAR families, and are named according to the thickening-purpose final-product viscosity which does not reflect the real composition. Consequently, this can limit their use in a clinical applications. To address this issue, six commercial λ-CARs were compared and differences in their physiochemical properties were analyzed and shown. Then, a H2O2-assisted depolymerization was applied to each commercial source, and number- and weight-averaged molar masses (Mn and Mw) and sulfation degree (DS) of the λ-COs produced over time were determined. By adjusting the depolymerization time for each product, almost comparable λ-CO formulations could be obtained in terms of molar masses and DS, which ranged within previously reported values suitable for antitumor properties. However, when the anti-HPSE activity of these new λ-COs was screened, small changes that could not be attributed only to their small length or DS changes between them were found, suggesting a role of other features, such as differences in the initial mixture composition. Further structural MS and NMR analysis revealed qualitative and semi-quantitative differences between the molecular species, especially in the proportion of the anti-HPSE λ-type, other CARs types and adjuvants, and it also showed that H2O2-based hydrolysis induced sugar degradation. Finally, when the effects of λ-COs were assessed in an in vitro migration cell-based model, they seemed more related to the proportion of other CAR types in the formulation than to their λ-type-dependent anti-HPSE activity.

Iron Oxide Nanoparticles Functionalized with Fucoidan: a Potential Theranostic Nanotool for Hepatocellular Carcinoma.

Fucoidan is a natural sulfated polysaccharide with a large range of biological activities including anticancer and anti-oxidation activities. Hepatocellular carcinoma is the fourth most common aggressive cancer type. The aim of this study was to investigate the bioactivity of free fucoidan versus its vectorization using nanoparticles (NPs) in human hepatoma cells, Huh-7. Iron oxide NPs were functionalized with fucoidan by a one-step surface complexation. NP cellular uptake was quantified by magnetic measurement at various extracellular iron concentrations. Cell invasion and migration were reduced with NPs while free fucoidan increases these events at low fucoidan concentration (≤0.5 µM). Concomitantly, a high decrease of reactive oxygen species production related with a decrease of the matrix metalloproteinase-9 activity and an increase of its expression was observed with NPs compared to free fucoidan. A proteomic analysis evidenced that some fucoidan regulated proteins appeared, which were related to protein synthesis, N-glycan processing, and cellular stress. To our knowledge, this is the first study which reveals such activity induced by fucoidan. These results pave the way for USPIO-fucoidan-NPs as potential theranostic nanotools for hepatocellular carcinoma treatment.

miR-126-3p is essential for CXCL12-induced angiogenesis.

Atherosclerosis, in the ultimate stage of cardiovascular diseases, causes an obstruction of vessels leading to ischemia and finally to necrosis. To restore vascularization and tissue regeneration, stimulation of angiogenesis is necessary. Chemokines and microRNAs (miR) were studied as pro-angiogenic agents. We analysed the miR-126/CXCL12 axis and compared impacts of both miR-126-3p and miR-126-5p strands effects in CXCL12-induced angiogenesis. Indeed, the two strands of miR-126 were previously shown to be active but were never compared together in the same experimental conditions regarding their differential functions in angiogenesis. In this study, we analysed the 2D-angiogenesis and the migration assays in HUVEC in vitro and in rat's aortic rings ex vivo, both transfected with premiR-126-3p/-5p or antimiR-126-3p/-5p strands and stimulated with CXCL12. First, we showed that CXCL12 had pro-angiogenic effects in vitro and ex vivo associated with overexpression of miR-126-3p in HUVEC and rat's aortas. Second, we showed that 2D-angiogenesis and migration induced by CXCL12 was abolished in vitro and ex vivo after miR-126-3p inhibition. Finally, we observed that SPRED-1 (one of miR-126-3p targets) was inhibited after CXCL12 treatment in HUVEC leading to improvement of CXCL12 pro-angiogenic potential in vitro. Our results proved for the first time: 1-the role of CXCL12 in modulation of miR-126 expression; 2-the involvement of miR-126 in CXCL12 pro-angiogenic effects; 3-the involvement of SPRED-1 in angiogenesis induced by miR-126/CXCL12 axis.

NADPH oxidase depletion in neutrophils from patients with cirrhosis and restoration via toll-like receptor 7/8 activation.

OBJECTIVE: Cirrhosis downregulates phagocyte oxidant production via their antibacterial superoxide-generating system, NADPH oxidase (NOX2) and increases patients' susceptibility to infection and mortality rate. To explore novel biochemical parameters that explain susceptibility to infections, we investigated the expression of NOX2 and partners in neutrophils of patients with severe alcoholic cirrhosis and have provided a novel approach to restore superoxide production capacity in patients' neutrophils and blood. DESIGN: Neutrophils were isolated from patients with decompensated alcoholic cirrhosis. NOX2 activity was assessed after stimulation of purified neutrophils or whole blood with the bacterial-derived peptide fMet-Leu-Phe. The expression of NOX2 and partners was studied by western blot analysis, flow cytometry and reverse transcription-PCR. RESULTS: The impaired superoxide production by patients' neutrophils was associated with a severe deficient expression of the NADPH oxidase catalytic core flavocytochrome-b558 (gp91 phox /NOX2 and p22 phox ), its cytosolic partner p47 phox but not p67 phox . NOX2 expression decreased rapidly by protein degradation involving elastase released during degranulation of healthy neutrophils stimulated with fMet-Leu-Phe, or highly present in patients' plasma. Interestingly, the deficient superoxide production was reversed by treatment of patients' neutrophils and whole blood with toll-like receptor 7/8 (TLR7/8) agonists. This treatment stimulated a rapid NOX2 transcription and translation through a process involving mammalian target of rapamycin (mTOR) whose expression was also deficient in patients' neutrophils. NOX2 expression was also increased by the TLR4 agonist lipopolysaccharide but with only a modest improvement of reactive oxygen species production. CONCLUSION: Impairment of neutrophil oxidants production in alcoholic cirrhosis is associated with NOX2 degradation and deficient mTOR-dependent translational machinery. The NOX2 depletion can be reversed via TRL7/8 activation and might be used to restore antimicrobial responses of immunocompromised patients.

Identification of a Pro-Angiogenic Potential and Cellular Uptake Mechanism of a LMW Highly Sulfated Fraction of Fucoidan from Ascophyllum nodosum.

Herein we investigate the structure/function relationships of fucoidans from Ascophyllum nodosum to analyze their pro-angiogenic effect and cellular uptake in native and glycosaminoglycan-free (GAG-free) human endothelial cells (HUVECs). Fucoidans are marine sulfated polysaccharides, which act as glycosaminoglycans mimetics. We hypothesized that the size and sulfation rate of fucoidans influence their ability to induce pro-angiogenic processes independently of GAGs. We collected two fractions of fucoidans, Low and Medium Molecular Weight Fucoidan (LMWF and MMWF, respectively) by size exclusion chromatography and characterized their composition (sulfate, fucose and uronic acid) by colorimetric measurement and Raman and FT-IR spectroscopy. The high affinities of fractionated fucoidans to heparin binding proteins were confirmed by Surface Plasmon Resonance. We evidenced that LMWF has a higher pro-angiogenic (2D-angiogenesis on Matrigel) and pro-migratory (Boyden chamber) potential on HUVECs, compared to MMWF. Interestingly, in a GAG-free HUVECs model, LMWF kept a pro-angiogenic potential. Finally, to evaluate the association of LMWF-induced biological effects and its cellular uptake, we analyzed by confocal microscopy the GAGs involvement in the internalization of a fluorescent LMWF. The fluorescent LMWF was mainly internalized through HUVEC clathrin-dependent endocytosis in which GAGs were partially involved. In conclusion, a better characterization of the relationships between the fucoidan structure and its pro-angiogenic potential in GAG-free endothelial cells was required to identify an adapted fucoidan to enhance vascular repair in ischemia.

Heparanase and Syndecan-4 Are Involved in Low Molecular Weight Fucoidan-Induced Angiogenesis.

Induction of angiogenesis is a potential treatment for chronic ischemia. Low molecular weight fucoidan (LMWF), the sulfated polysaccharide from brown seaweeds, has been shown to promote revascularization in a rat limb ischemia, increasing angiogenesis in vivo. We investigated the potential role of two heparan sulfate (HS) metabolism enzymes, exostosin-2 (EXT2) and heparanase (HPSE), and of two HS-membrane proteoglycans, syndecan-1 and -4 (SDC-1 and SDC-4), in LMWF induced angiogenesis. Our results showed that LMWF increases human vascular endothelial cell (HUVEC) migration and angiogenesis in vitro. We report that the expression and activity of the HS-degrading HPSE was increased after LMWF treatment. The phenotypic tests of LMWF-treated and EXT2- or HPSE-siRNA-transfected cells indicated that EXT2 or HPSE expression significantly affect the proangiogenic potential of LMWF. In addition, LMWF increased SDC-1, but decreased SDC-4 expressions. The effect of LMWF depends on SDC-4 expression. Silencing EXT2 or HPSE leads to an increased expression of SDC-4, providing the evidence that EXT2 and HPSE regulate the SDC-4 expression. Altogether, these data indicate that EXT2, HPSE, and SDC-4 are involved in the proangiogenic effects of LMWF, suggesting that the HS metabolism changes linked to LMWF-induced angiogenesis offer the opportunity for new therapeutic strategies of ischemic diseases.

New prospects in the roles of the C-terminal domains of VEGF-A and their cooperation for ligand binding, cellular signaling and vessels formation.

VEGF-A is a crucial growth factor for blood vessel homeostasis and pathological angiogenesis. Due to alternative splicing of its pre-mRNA, VEGF-A is produced under several isoforms characterized by the combination of their C-terminal domains, which determines their respective structure, availability and affinity for co-receptors. As controversies still exist about the specific roles of these exon-encoded domains, we systematically compared the properties of eight natural and artificial variants containing the domains encoded by exons 1-4 and various combinations of the domains encoded by exons 5, 7 and 8a or 8b. All the variants (VEGF111a, VEGF111b, VEGF121a, VEGF121b, VEGF155a, VEGF155b, VEGF165a, VEGF165b) have a similar affinity for VEGF-R2, as determined by Surface plasmon resonance analyses. They strongly differ however in terms of binding to neuropilin-1 and heparin/heparan sulfate proteoglycans. Data indicate that the 6 amino acids encoded by exon 8a must be present and cooperate with those of exons 5 or 7 for efficient binding, which was confirmed in cell culture models. We further showed that VEGF165b has inhibitory effects in vitro, as previously reported, but that the shortest VEGF variant possessing also the 6 amino acids encoded by exon 8b (VEGF111b) is remarkably proangiogenic, demonstrating the critical importance of domain interactions for defining the VEGF properties. The number, size and localization of newly formed blood vessels in a model of tumour angiogenesis strongly depend also on the C-terminal domain composition, suggesting that association of several VEGF isoforms may be more efficient for treating ischemic diseases than the use of any single variant.

TIEG1-null tenocytes display age-dependent differences in their gene expression, adhesion, spreading and proliferation properties.

The remodeling of extracellular matrix is a crucial mechanism in tendon development and the proliferation of fibroblasts is a key factor in this process. The purpose of this study was to further elucidate the role of TIEG1 in mediating important tenocyte properties throughout the aging process. Wildtype and TIEG1 knockout tenocytes adhesion, spreading and proliferation were characterized on different substrates (fibronectin, collagen type I, gelatin and laminin) and the expression levels of various genes known to be involved with tendon development were analyzed by RT-PCR. The experiments revealed age-dependent and substrate-dependent properties for both wildtype and TIEG1 knockout tenocytes. Taken together, our results indicate an important role for TIEG1 in regulating tenocytes adhesion, spreading, and proliferation throughout the aging process. Understanding the basic mechanisms of TIEG1 in tenocytes may provide valuable information for treating multiple tendon disorders.

Tumor cell/endothelial cell tight contact upregulates endothelial adhesion molecule expression mediated by NFkappaB: differential role of the shear stress.

Cancer metastasis is a multistep process involving cell-cell interactions, but little is known about the adhesive interactions and signaling events during extravasation of tumor cells (TCs). In this study, cell adhesion molecule (CAM) expression was investigated using an in vitro assay, in which TCs were seeded onto an endothelial cell (ECs) monolayer and cocultured during 5 h. Flow cytometry, confocal microscopy as well as western blot analysis indicated that endothelial ICAM-1 (Inter Cellular Adhesion Molecule-1), VCAM-1 (Vascular Adhesion Molecule-1) and E-selectin were up-regulated after TC-EC coculture, whereas no change was observed for CAMs expression in tumor cells. This increased CAMs expression required tight contact between TCs and ECs. Incubation of ECs with the pyrrolidine-dithiocarbamate NFkappaB inhibitor prior to coculture, fully prevented coculture-induced expression of endothelial CAMs. Using specific blocking antibodies we showed an implication of ICAM-1 and VCAM-1 for TCs extravasation and VCAM-1 for adhesion. Moreover, fluid flow experiments revealed that high shear stress totally abolished coculture-induced as well as TNFalpha-induced CAMs over-expression. This study suggests that TCs could act as a potent inflammatory stimulus on ECs by inducing CAMs expression via NFkappaB activation, and that this action can be modulated by shear stress.

Syndecan-1 and syndecan-4 are involved in RANTES/CCL5-induced migration and invasion of human hepatoma cells.

BACKGROUND: We previously demonstrated that the CC-chemokine Regulated upon Activation, Normal T cell Expressed and Secreted (RANTES)/CCL5 exerts pro-tumoral effects on human hepatoma Huh7 cells through its G protein-coupled receptor, CCR1. Glycosaminoglycans play major roles in these biological events. METHODS: In the present study, we explored 1/ the signalling pathways underlying RANTES/CCL5-mediated hepatoma cell migration or invasion by the use of specific pharmacological inhibitors, 2/ the role of RANTES/CCL5 oligomerization in these effects by using a dimeric RANTES/CCL5, 3/ the possible involvement of two membrane heparan sulfate proteoglycans, syndecan-1 (SDC-1) and syndecan-4 (SDC-4) in RANTES/CCL5-induced cell chemotaxis and spreading by pre-incubating cells with specific antibodies or by reducing SDC-1 or -4 expression by RNA interference. RESULTS AND CONCLUSION: The present data suggest that focal adhesion kinase phosphorylation, phosphoinositide 3-kinase-, mitogen-activated protein kinase- and Rho kinase activations are involved in RANTES/CCL5 pro-tumoral effects on Huh7 cells. Interference with oligomerization of the chemokine reduced RANTES/CCL5-mediated cell chemotaxis. This study also indicates that SDC-1 and -4 may be required for HepG2, Hep3B and Huh7 human hepatoma cell migration, invasion or spreading induced by the chemokine. These results also further demonstrate the involvement of glycosaminoglycans as the glycosaminoglycan-binding deficient RANTES/CCL5 variant, in which arginine 47 was replaced by lysine, was devoid of effect. GENERAL SIGNIFICANCE: The modulation of RANTES/CCL5-mediated cellular effects by targeting the chemokine-syndecan interaction could represent a new therapeutic approach for hepatocellular carcinoma.

Monocyte chemoattractant protein-1 (MCP-1)/CCL2 secreted by hepatic myofibroblasts promotes migration and invasion of human hepatoma cells.

The aim of our study was to investigate whether myofibroblasts and the chemokine monocyte chemoattractant protein-1 (MCP-1)/CCL2 may play a role in hepatocellular carcinoma progression. We observed that hepatic myofibroblast LI90 cells express MCP-1/CCL2 mRNA and secrete this chemokine. Moreover, myofibroblast LI90 cell-conditioned medium (LI90-CM) induces human hepatoma Huh7 cell migration and invasion. These effects are strongly reduced when a MCP-1/CCL2-depleted LI90-CM was used. We showed that MCP-1/CCL2 induces Huh7 cell migration and invasion through its G-protein-coupled receptor CCR2 and, to a lesser extent, through CCR1 only at high MCP-1/CCL2 concentrations. MCP-1/CCL2's chemotactic activities rely on tyrosine phosphorylation of focal adhesion components and depend on matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, we observed that Huh7 cell migration and invasion induced by the chemokine are strongly inhibited by heparin, by beta-D-xyloside treatment of cells and by anti-syndecan-1 and -4 antibodies. Finally, we developed a 3-dimensional coculture model of myofibroblast LI90 and Huh7 cells and demonstrated that MCP-1/CCL2 and its membrane partners, CCR1 and CCR2, may be involved in the formation of mixed hepatoma-myofibroblast spheroids. In conclusion, our data show that human liver myofibroblasts act on hepatoma cells in a paracrine manner to increase their invasiveness and suggest that myofibroblast-derived MCP-1/CCL2 could be involved in the pathogenesis of hepatocellular carcinoma.

Glycosaminoglycan mimetics inhibit SDF-1/CXCL12-mediated migration and invasion of human hepatoma cells.

We have recently reported that the CXC-chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 induces proliferation, migration, and invasion of the Huh7 human hepatoma cells through its G-protein-coupled receptor CXCR4 and that glycosaminoglycans (GAGs) are involved in these events. Here, we demonstrate by surface plasmon resonance that the chemokine binds to GAG mimetics obtained by grafting carboxylate, sulfate or acetate groups onto a dextran backbone. We also demonstrate that chemically modified dextrans inhibit SDF-1/CXCL12-mediated in vitro chemotaxis and anchorage-independent cell growth in a dose-dependent manner. The binding of GAG mimetics to the chemokine and their effects in modulating the SDF-1/CXCL12 biological activities are mainly related to the presence of sulfate groups. Furthermore, the mRNA expression of enzymes involved in heparan sulfate biosynthesis, such as exostosin-1 and -2 or N-deacetylase N-sulfotransferases remained unchanged, but heparanase mRNA and protein expressions in Huh7 cells were decreased upon GAG mimetic treatment. Moreover, decreasing heparanase-1 mRNA levels by RNA interference significantly reduced SDF-1/CXCL12-induced extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. Therefore, we suggest that GAG mimetic effects on SDF-1/CXCL12-mediated hepatoma cell chemotaxis may rely on decreased heparanase expression, which impairs SDF-1/CXCL12's signaling. Altogether, these data suggest that GAG mimetics may compete with cellular heparan sulfate chains for the binding to SDF-1/CXCL12 and may affect heparanase expression, leading to reduced SDF-1/CXCL12 mediated in vitro chemotaxis and growth of hepatoma cells.

TIEG1-NULL OSTEOCYTES DISPLAY DEFECTS IN THEIR MORPHOLOGY, DENSITY AND SURROUNDING BONE MATRIX.

Through the development of TGFß-inducible early gene-1 (TIEG1) knockout (KO) mice, we have demonstrated that TIEG1 plays an important role in osteoblast-mediated bone mineralization, and in bone resistance to mechanical strain. To further investigate the influence of TIEG1 in skeletal maintenance, osteocytes were analyzed by transmission electron microscopy using TIEG1 KO and wild-type mouse femurs at one, three and eight months of age. The results revealed an age-dependent change in osteocyte surface and density, suggesting a role for TIEG1 in osteocyte development. Moreover, there was a decrease in the amount of hypomineralized bone matrix surrounding the osteocytes in TIEG1 KO mice relative to wild-type controls. While little is known about the function or importance of this hypomineralized bone matrix immediately adjacent to osteocytes, this study reveals significant differences in this bone microenvironment and suggests that osteocyte function may be compromised in the absence of TIEG1 expression.

Glycosaminoglycans and their synthetic mimetics inhibit RANTES-induced migration and invasion of human hepatoma cells.

The CC-chemokine regulated on activation, normal T-cell expressed, and presumably secreted (RANTES)/CCL5 mediates its biological activities through activation of G protein-coupled receptors, CCR1, CCR3, or CCR5, and binds to glycosaminoglycans. This study was undertaken to investigate whether this chemokine is involved in hepatoma cell migration or invasion and to modulate these effects in vitro by the use of glycosaminoglycan mimetics. We show that the human hepatoma Huh7 and Hep3B cells express RANTES/CCL5 G protein-coupled receptor CCR1 but not CCR3 nor CCR5. RANTES/CCL5 binding to these cells depends on CCR1 and glycosaminoglycans. Moreover, RANTES/CCL5 strongly stimulates the migration and the invasion of Huh7 cells and to a lesser extent that of Hep3B cells. RANTES/CCL5 also stimulates the tyrosine phosphorylation of focal adhesion kinase and activates matrix metalloproteinase-9 in Huh7 hepatoma cells, resulting in increased invasion of these cells. The fact that RANTES/CCL5-induced migration and invasion of Huh7 cells are both strongly inhibited by anti-CCR1 antibodies and heparin, as well as by beta-d-xyloside treatment of the cells, suggests that CCR1 and glycosaminoglycans are involved in these events. We then show by surface plasmon resonance that synthetic glycosaminoglycan mimetics, OTR4120 or OTR4131, directly bind to RANTES/CCL5. The preincubation of the chemokine with each of these mimetics strongly inhibited RANTES-induced migration and invasion of Huh7 cells. Therefore, targeting the RANTES-glycosaminoglycan interaction could be a new therapeutic approach for human hepatocellular carcinoma.

Morphological analysis of tumor cell/endothelial cell interactions under shear flow.

In the process of hematogenous cancer metastasis, tumor cells (TCs) must shed into the blood stream, survive in the blood circulation, migrate through the vascular endothelium (extravasation) and proliferate in the target organs. However, the precise mechanisms by which TCs penetrate the endothelial cell (EC) junctions remain one of the least understood aspects of TC extravasation. This question has generally been addressed under static conditions, despite the important role of flow induced mechanical stress on the circulating cell-endothelium interactions. Moreover, flow studies were generally focused on transient or firm adhesion steps of TC-EC interactions and did not consider TCs spreading or extravasation. In this paper, we used a parallel-plate flow chamber to investigate TC-EC interactions under flow conditions. An EC monolayer was cultured on the lower plate of the flow chamber to model the endothelial barrier. Circulating TCs were introduced into the flow channel under a well-defined flow field and TC cell shape changes on the EC monolayer were followed in vitro with live phase contrast and fluorescence microscopy. Two spreading patterns were observed: radial spreading which corresponds to TC extravasation, and axial spreading where TCs formed a mosaic TC-EC monolayer. By investigating the changes in area and minor/major aspect ratio, we have established a simple quantitative basis for comparing spreading modes under various shear stresses. Contrary to radial spreading, the extent of axial spreading was increased by shear stress.

miR-126 Is Involved in Vascular Remodeling under Laminar Shear Stress.

Morphology and changes in gene expression of vascular endothelium are mainly due to shear stress and inflammation. Cell phenotype modulation has been clearly demonstrated to be controlled by small noncoding micro-RNAs (miRNAs). This study focused on the effect of laminar shear stress (LSS) on human endothelial cells (HUVECs), with an emphasis on the role of miRNA-126 (miR-126). Exposure of HUVECs in vitro to LSS modified the shape of HUVECs and concomitantly regulated the expression of miR-126, vascular cell adhesion molecule 1 (VCAM-1), and syndecan-4 (SDC-4). A significant upregulation of miR-126 during long-term exposure to flow was shown. Interestingly, LSS enhanced SDC-4 expression on the HUVEC membranes. Overexpression of miR-126 in HUVECs decreased the levels of targets stromal cell-derived factor-1 SDF-1/CXCL12 and VCAM-1 but increased the expression of RGS16, CXCR4, and SDC-4. No significant difference in terms of cell proliferation and apoptosis was observed between scramble, anti-miR-126, and pre-miR-126 transfected HUVECs. In Apo-E KO/CKD mice aortas expressing a high level of miR-126, SDC-4 was concomitantly increased. In conclusion, our results suggest that miR-126 (i) is overexpressed by long-term LSS, (ii) has a role in up- and downregulation of genes involved in atherosclerosis, and (iii) affects SDC-4 expression.

Fucoidan in a 3D scaffold interacts with vascular endothelial growth factor and promotes neovascularization in mice.

The aim of this study was to functionalize 3D porous cross-linked scaffolds with natural non-animal sulfated polysaccharide fucoidans in order to allow a delivery of vascular endothelial growth factor (VEGF) and potentiate its angiogenic activity. Microporous (20 µm) and macroporous (200 µm) scaffolds were functionalized with low, medium, or high molecular weight fucoidans (named LMWF, MMWF, and HMWF, respectively). In vitro, addition of fucoidans promoted endothelial progenitor cells proliferation in both micro- and macroporous scaffolds. While control scaffolds without fucoidans loaded with VEGF165 (100 ng) showed a fast burst release in PBS during the first 24 h, MMWF significantly reduced the VEGF165 release (p < 0.001). Surface plasmon resonance experiments confirmed a direct interaction between MMWF and VEGF165, characterized by an affinity K D (K d/K a) of 1 × 10(-9) M. In a subcutaneous angiogenesis model in mice, fucoidan functionalized scaffolds showed a more intense vascularization response than control groups. Expression of isolectin-B4 and α-smooth muscle actin, as well as confinement of erythrocytes, demonstrated the neoformed blood vessels functionality. There was a significant difference in neovessel area and neovessel density between MMWF scaffolds or VEGF165 scaffolds and MMWF+VEGF165 scaffolds (p < 0.001 for all cases). Here, we demonstrate that fucoidan sequesters VEGF165 and delivers biological cues promoting angiogenesis. In conclusion, this study shows that hydrogels functionalized with fucoidan can direct the formation of mature vasculature through a local release of VEGF165 and can be a useful tool in ischemic tissues to guide therapeutic angiogenesis.

Ultrasmall superparamagnetic iron oxide nanoparticles coated with fucoidan for molecular MRI of intraluminal thrombus.

AIM: We have designed ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles associated with fucoidan (USPOI-FUCO), a natural sulfated polysaccharide with high affinity for activated platelets, to visualize by MRI arterial thrombi. MATERIALS & METHODS: USPIOs were prepared and sizes, zeta-potentials and relaxivities were measured. Elastase perfusion in the infrarenal aorta of Wistar rats induced intraluminal thrombus. They were scanned on 4.7 T MRI before and after injection of USPIO-FUCO or USPIO coated with anionic dextran. RESULTS: Surface plasmon resonance evidenced that fucoidan and USPIO-FUCO bind in vitro to immobilized P-selectin. All intraluminal hyposignals detected by MRI after injection of USPIO-FUCO on animals (13 out of 13) were correlated by histology with thrombi, whereas none could be identified with control USPIOs (0 out of 7). No signal was seen in absence of thrombus. Thrombi by MRI were correlated with P-selectin immunostaining and USPIO detection by electron microscopy. CONCLUSION: In vivo thrombi can thus be evidenced by MRI with USPIO-FUCO.

RANTES/CCL5 mediated-biological effects depend on the syndecan-4/PKCα signaling pathway.

The perpetuation of angiogenesis is involved in certain chronic inflammatory diseases. The accelerated neovascularisation may result from an inflammatory status with a response of both endothelial cells and monocytes to inflammatory mediators such as chemokines. We have previously described in vitro and in vivo the pro-angiogenic effects of the chemokine Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES)/CCL5. The effects of RANTES/CCL5 may be related to its binding to G protein-coupled receptors and to proteoglycans such as syndecan-1 and -4. The aim of this study was to evaluate the functionality of syndecan-4 as a co-receptor of RANTES/CCL5 by the use of mutated syndecan-4 constructs. Our data demonstrate that site-directed mutations in syndecan-4 modify RANTES/CCL5 biological activities in endothelial cells. The SDC4S179A mutant, associated with an induced protein kinase C (PKC)α activation, leads to higher RANTES/CCL5 pro-angiogenic effects, whereas the SDC4L188QQ and the SDC4A198del mutants, leading to lower phosphatidylinositol 4,5-bisphosphate (PIP2) binding or to lower PDZ protein binding respectively, are associated with reduced RANTES/CCL5 cellular effects. Moreover, our data highlight that the intracellular domain of SDC-4 is involved in RANTES/CCL5-induced activation of the PKCα signaling pathway and biological effect. As RANTES/CCL5 is involved in various physiopathological processes, the development of a new therapeutic strategy may be reliant on the mechanism by which RANTES/CCL5 exerts its biological activities, for example by targeting the binding of the chemokine to its proteoglycan receptor.

Abdominal aortic aneurysms targeted by functionalized polysaccharide microparticles: a new tool for SPECT imaging.

Aneurysm diagnostic is nowadays limited by the lack of technology that enables early detection and rupture risk prediction. New non invasive tools for molecular imaging are still required. In the present study, we present an innovative SPECT diagnostic tool for abdominal aortic aneurysm (AAA) produced from injectable polysaccharide microparticles radiolabeled with technetium 99m ((99m)Tc) and functionalized with fucoidan, a sulfated polysaccharide with the ability to target P-Selectin. P-Selectin is a cell adhesion molecule expressed on activated endothelial cells and platelets which can be found in the thrombus of aneurysms, as well as in other vascular pathologies. Microparticles with a maximum hydrodynamic diameter of 4 µm were obtained by crosslinking the polysaccharides dextran and pullulan. They were functionalized with fucoidan. In vitro interactions with human activated platelets were assessed by flow cytometry that demonstrated a specific affinity of fucoidan functionalized microparticles for P-Selectin expressed by activated platelets. For in vivo AAA imaging, microparticles were radiolabeled with (99m)Tc and intravenously injected into healthy and AAA rats obtained by elastase perfusion through the aorta wall. Animals were scanned by SPECT imaging. A strong contrast enhancement located in the abdominal aorta of AAA rats was obtained, while no signal was obtained in healthy rats or in AAA rats after injection of non-functionalized control microparticles. Histological studies revealed that functionalized radiolabeled polysaccharide microparticles were localized in the AAA wall, in the same location where P-Selectin was expressed. These microparticles therefore constitute a promising SPECT imaging tool for AAA and potentially for other vascular diseases characterized by P-Selectin expression. Future work will focus on validating the efficiency of the microparticles to diagnose these other pathologies and the different stages of AAA. Incorporation of a therapeutic molecule is also considered.