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.

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.

Optimized multimodal nanoplatforms for targeting α(v)ß3 integrins.

Magnetic Resonance Imaging (MRI) using contrast agents is a very powerful technique for diagnosis in clinical medicine and biomedical research. The synthesis of ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles targeting αvß3 integrins and acting as new MRI contrast agents seems to be a promising way for cancer diagnosis. Indeed, it is well established that αvß3 integrin plays a key role in tumor angiogenesis acting like a receptor for the extracellular matrix proteins like vitronectin, fibronectin through the arginine-glycine-aspartic acid (RGD) sequence. Up-regulation of αvß3 has been found to be associated with a wide range of cancers, making it a broad-spectrum tumor-marker. In this study, USPIO nanocrystals were synthesized and surface passivated with caffeic acid. The large number of the carboxylic acid functions at the outer surface of the nanoplatforms was used for the covalent coupling of Rhodamine123, polyethylene glycol (PEG) and cyclic RGD. Soluble carbodiimide (EDC) and N-hydroxysuccinimide (NHS) were used to crosslink carboxylic acid with the amino group of the ligands. We examined the design of the nanoplatforms with each individual entity and then the combination of two and three of them. Several methods were used to characterize the nanoparticle surface functionalization and the magnetic properties of these contrast agents were studied using a 1.5 T clinical MRI scanner. The affinity towards integrins was evidenced by surface plasmon resonance and solid-phase receptor-binding assay.

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.

Inhibition of VEGF expression in A431 and MDA-MB-231 tumour cells by cationic lipid-mediated siRNA delivery.

In order to promote siRNA transfer in tumour cells, we used an original cationic lipid, synthesized in our laboratory, dimethyl-hydroxyethyl-aminopropane-carbamoyl-cholesterol (DMHAPC-Chol). Liposomes were prepared from this lipid and dioleoylphosphatidylethanolamine (DOPE) in equimolar proportion. Its transfecting capacity was evaluated using ELISA, cell cytometry, and RT-PCR in estimating the silencing effect of VEGF siRNA. This liposome efficiently delivered VEGF siRNA in two human cancer cell lines abundantly secreting VEGF, A431 and MDA-MB-231. Results showed that 50 nM of VEGF siRNA carried by DMHAPC-Chol/DOPE liposomes already silenced more than 90% of VEGF in these cells. A comparative study with two commercial carriers indicated that the inhibition induced by VEGF siRNA transported by cationic DMHAPC-Chol/DOPE liposomes was comparable to that induced by INTERFERin and better than lipofectamine 2000. Moreover, a transfection by a GFP plasmid followed by a GFP siRNA showed that DMHAPC-Chol/DOPE liposomes compared to lipofectamine were less efficient for plasmid but better for siRNA transport. Following one of our previous works concerning cell delivery of plasmid ( Percot et al., 2004 ), the main interest of results presented here resides in the double potential of DMHAPC-Chol/DOPE liposomes to deliver little-sized siRNA as well as large nucleic acids in cells.

The mechanical behavior of mutant K14-R125P keratin bundles and networks in NEB-1 keratinocytes.

Epidermolysis bullosa simplex (EBS) is an inherited skin-blistering disease that is caused by dominant mutations in the genes for keratin K5 or K14 proteins. While the link between keratin mutations and keratinocyte fragility in EBS patients is clear, the exact biophysical mechanisms underlying cell fragility are not known. In this study, we tested the hypotheses that mutant K14-R125P filaments and/or networks in human keratinocytes are mechanically defective in their response to large-scale deformations. We found that mutant filaments and networks exhibit no obvious defects when subjected to large uniaxial strains and have no negative effects on the ability of human keratinocytes to survive large strains. We also found that the expression of mutant K14-R125P protein has no effect on the morphology of the F-actin or microtubule networks or their responses to large strains. Disassembly of the F-actin network with Latrunculin A unexpectedly led to a marked decrease in stretch-induced necrosis in both WT and mutant cells. Overall, our results contradict the hypotheses that EBS mutant keratin filaments and/or networks are mechanically defective. We suggest that future studies should test the alternative hypothesis that keratinocytes in EBS cells are fragile because they possess a sparser keratin network.

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.

Low molecular weight fucoidan prevents intimal hyperplasia in rat injured thoracic aorta through the modulation of matrix metalloproteinase-2 expression.

The therapeutic potential of low molecular-weight fucoidan (LMWF), a sulfated polysaccharide extracted from brown seaweed was investigated on vascular smooth muscle cell (VSMC) and human vascular endothelial cell (HUV-EC-C) proliferation and migration in vitro and in a rat model of intimal hyperplasia. Sprague-Dawley rats were subjected to balloon injury in the thoracic aorta followed by two weeks' treatment with either LMWF (5mg/kg/day) or vehicle. Morphological analysis and proliferating cell nuclear antigen immunostaining at day 14 indicated that LMWF prevented intimal hyperplasia in rat thoracic aorta as compared with vehicle (neo-intima area, 3±0.50mm(2) versus 5±0.30mm(2), P<0.01). In situ zymography showed that LMWF significantly decreased the activity of matrix metalloproteinase (MMP)-2 in the neo-intima compared to vehicle. The in vitro study demonstrated that 10µg/ml LMWF increased HUV-EC-C migration by 45±5% but reduced VSMC migration by 40±3%. LMWF also increased MMP-2 mRNA expression in HUV-EC-Cs and reduced it in VSMCs. MMP-2 level in the conditioned medium from cells incubated with 10µg/ml LMWF was 5.4-fold higher in HUV-EC-Cs, but 6-fold lower in VSMCs than in untreated control cells. Furthermore, decreasing MMP-2 expression in HUV-EC-Cs or VSMCs by RNA interference resulted in reduced LMWF-induced effects on cell migration. In conclusion, LMWF increased HUV-EC-C migration and decreased VSMC migration in vitro. In vivo, this natural compound reduced the intimal hyperplasia in the rat aortic wall after balloon injury. Therefore, LMWF could be of interest for the prevention of intimal hyperplasia.

Molecular structure of tail tendon fibers in TIEG1 knockout mice using synchrotron diffraction technology.

The purpose of this study was to characterize the effect of TIEG1 on the molecular structure of collagen within tail tendon fibers using 3-mo-old female C57BL/6 wild-type (WT) and TIEG1 KO mice. Synchrotron X-ray microdiffraction experiments were carried out on single tendon fibers extracted from the WT and TIEG1 KO dorsal tail tendon. The fibers were scanned in the radial direction, and X-ray patterns were obtained. From these patterns, the meridional direction was analyzed through X-ray intensity profile. In addition, collagen content was investigated using hydroxyproline assays, and qualitative real-time PCR experiments were performed on RNA isolated from fibroblasts to examine specific gene expression changes. The results showed different X-ray diffraction patterns between WT and TIEG1 KO tendon fibers, indicating a disorganization of the collagen structure for the TIEG1 KO compared with WT mice. Furthermore, the analyses of the X-ray intensity profiles exhibited a higher (23 A) period of collagen for the TIEG1 KO compared with the WT mice. The results of the hydroxyproline assays revealed a significant decrease in the TIEG1 KO compared with WT mice, leading to a decrease in the total amount of collagen present within the TIEG1 KO tendons. Moreover, qualitative real-time PCR results showed differences in the expression profiles of specific genes known to play important roles in tendon fiber development. These data further elucidate the role of TIEG1 on tendon structure and could explain the previous defects in the structure-function relationship found for TIEG1 KO tendon fibers.

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.

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.