Olivier Oudar

RANTES/CCL5-induced pro-angiogenic effects depend on CCR1, CCR5 and glycosaminoglycans.

Atherosclerosis involves angiogenesis and inflammation with the ability of endothelial cells and monocytes to respond to chemokines. We addressed here by in vitro and in vivo approaches, the role of the chemokine Regulated on Activation, Normal T Cell Expressed and Secreted (RANTES)/CCL5 on angiogenesis through its receptors CCR1, CCR5, syndecan-1 (SDC-1), syndecan-4 (SDC-4) and CD-44. Our data demonstrate that RANTES/CCL5 is pro-angiogenic in a rat subcutaneous model. This RANTES/CCL5-activity may be related to the in vitro promotion of endothelial cell migration, spreading and neo-vessel formation. RANTES/CCL5-mediated angiogenesis depends at least partly on Vascular Endothelial Growth Factor (VEGF) secretion by endothelial cells, since this effect is decreased when endothelial cells are incubated with anti-VEGF receptor antibodies. RANTES/CCL5-induced chemotaxis is mediated by matrix metalloproteinase-9. We demonstrate that specific receptors of RANTES/CCL5 such as G protein-coupled receptors CCR1 and CCR5, and heparan sulfate proteoglycans, SDC-1, SDC-4 or CD-44, play a major role in RANTES/CCL5-induced angiogenic effects. By the use of two RANTES/CCL5 mutants, [E66A]-RANTES/CCL5 with impaired ability to oligomerize, and [44AANA47]-RANTES/CCL5 mutated in the main RANTES/CCL5-glycosaminoglycan (GAG) binding site, we demonstrate that chemokine oligomerization and binding to GAGs are essential in RANTES/CCL5-induced angiogenic effects. According to these results, new therapeutic strategies based on RANTES/CCL5 can be proposed for neo-angiogenesis after vascular injury. Mutants of RANTES/CCL5 may also represent an innovative approach to prevent the angiogenesis associated with the formation of atherosclerotic plaque.

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/CCL2s 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 β‐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.

Angiogenic properties of the chemokine RANTES/CCL5

Atherosclerosis is an inflammatory disease that is one of the leading causes of death in developed countries. This disease is defined by the formation of an atherosclerotic plaque, which is responsible for artery obstruction and affects the heart by causing myocardial infarction. The vascular wall is composed of three cell types and includes a monolayer of endothelial cells and is irrigated by a vasa vasorum. The formation of the vascular network from the vasa vasorum is a process involved in the destabilization of this plaque. Cellular and molecular approaches are studied by in vitro assay of activated endothelial cells and in in vivo models of neovascularization. Chemokines are a large family of small secreted proteins that have been shown to play a critical role in the regulation of angiogenesis during several pathophysiological processes such as ischaemia. Chemokines may exert their regulatory activity on angiogenesis directly by activating the vasa vasorum, or as a consequence of leucocyte infiltration through the endothelium, and/or by the induction of growth factor expression such as that of VEGF (vascular endothelial growth factor). The present review focuses on the angiogenic activity of the chemokines RANTES (regulated upon activation, normal T-cell expressed and secreted)/CCL5 (CC chemokine ligand 5). RANTES/CCL5 is released by many cell types such as platelets or smooth muscle cells. This chemokine interacts with GPCRs (G-protein-coupled receptors) and GAG (glycosaminoglycan) chains bound to HSPGs (heparan sulfate proteoglycans). Many studies have demonstrated, using RANTES/CCL5 mutated on their GAG or GPCR-binding sites, the involvement of these chemokines in angiogenic process. In the present review, we discuss two controversial roles of RANTES/CCL5 in the angiogenic process.

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.

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/CXCL12s 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.

Expression and regulation of intercellular adhesion molecule-1 (ICAM-1) in organotypic cultures of rat liver tissue

BACKGROUND/AIMS The objective of the present study was to analyze the expression and regulation of intercellular adhesion molecule-1 (ICAM-1) in organotypic cultures of rat liver slices, which preserve the normal microenvironment of liver cells. METHODS Rat liver slices were maintained in culture for 15 min to 24 h and examined for ICAM-1 expression by immunohistochemistry and Western blotting in basal conditions and after stimulation with 1000 IU/ml interferon-gamma (IFNgamma), 1000 IU/ml tumor necrosis factor-alpha (TNF alpha) and 50 microg/ml endotoxin. Immunohistochemical results were evaluated using a semiquantitative scoring system. RESULTS In uncultured slices, ICAM-1 was not detected on hepatocytes. In unstimulated liver slices maintained in organotypic culture, ICAM-1 was induced at the surface of scattered hepatocytes (score at 15 min, 0.33+/-0.47 and at 24 h, 1.17+/-0.69). After 4 h of stimulation, a significant increase in ICAM-1 expression by hepatocytes and adjacent sinusoidal cells, but not by intra-hepatic biliary epithelial cells, was observed for IFNgamma (score: 2.35+/-0.47) and endotoxin (score: 2.67+/-0.47), but not with TNF alpha (score: 0.66+/-0.47). After 24 h of stimulation, a further increase in the extent of ICAM-1 expression by hepatocytes was observed for IFNgamma (score: 3.67+/-0.47) and endotoxin (score: 4.0+/-0.0), and a significant overexpression of ICAM-1 by hepatocytes was detectable after treatment with TNF alpha (score: 3.67+/-0.47). CONCLUSIONS In rat liver organotypic cultures, TNF alpha, IFNgamma and endotoxin induce the expression of ICAM-1 in hepatocytes and adjacent sinusoidal endothelial cells, but not in portal tracts.

Spatial organization of three-dimensional cocultures of adriamycin-sensitive and -resistant human breast cancer MCF-7 cells

Genetic and cellular heterogeneity is one of mechanisms involved in increasing tumour aggressiveness during neoplastic progression. Development of drug‐resistant tumour cell subpopulations is a major problem in clinical oncology. Multi‐drug resistant tumour cells survive when exposed to cytotoxic agents. Here, we studied in a three‐dimensional (3D) coculture system, called “ex vivo nodules”, how drug‐resistant and sensitive tumour cells settle down in a 3D space. For this, we cocultured adriamycin‐sensitive (MCF‐7S) and ‐resistant (MCF‐7R) human breast cancer cells in long term nodules. We showed that both types of cells are able to grow separately or in coculture until five weeks in spheroidal forms. MCF‐7R cells did not loose their multi‐drug resistance when cultured in nodules as measured by RT‐PCR. Curiously, the exterior aspects of mixed (MCF‐7S/ MCF‐7R) nodules and MCF‐7R nodules were similar whereas MCF‐7S nodules were completely different. Nevertheless, morphologically these three nodule types were distinct, in particular in their density. Immunostaining showed that in mixed nodules, MCF‐7R cells were arranged at the periphery, whereas the MCF‐7S cells are in the central part of the nodules.

Synthesis and biological evaluation of new bisphosphonate-dextran conjugates targeting breast primary tumor.

Bisphosphonates (BPs) have interesting antitumor effects as well in vitro as in vivo, despite their poor bioavailability in the organism after oral ingestion. To overcome this problem and reduce drug doses and secondary effects, we report the chemical synthesis of new bioconjugates. They were built with a nitrogen-containing BP as the drug covalently coupled to the carboxymethyldextran. This polysaccharide was used as a carrier, in order to increase BP lifetime in bloodstream and to target tumor cells which have a strong affinity with dextran. The efficiency of our vectorization system was biologically proved in vitro and in vivo on mammalian carcinoma models in mice.

Secretion of Endolymph by the Isolated Frog Semicircular Canal

Secretion of endolymph is localized in some structures of the inner ear, namely the stria vascularis in the cochlea and the dark cells in the vestibule and in the lower vertebrate inner ear. In isolated semicircular canal it is possible to study separately the endolymphatic composition in the ampulla, which contains the dark cells, and in its non-ampullar part, which is devoid of these cells. Further, in vitro preparation of the semicircular canal provides access to both faces of the epithelium so that different agents can be applied separately to the apical or to the basolateral membranes of the epithelium. In this structure, the following results were obtained: i) in vitro, the semicircular canal secreted a K-rich, positively polarized fluid; ii) this fluid was secreted only in the ampulla of the semicircular canal; iii) the secretion of endolymph was dependent on basolateral Na+, K(+)-ATPase, inhibited by ouabain, and basolateral Na-K-Cl co-transporter, inhibited by bumetanide; iv) approximately 60% of luminal Na absorption occurred across a luminal Na channel inhibited by amiloride; v) the permeability of the paracellular pathway of the semicircular canal epithelium was 7.10(-7) cm/s. These results indicate that endolymph secretion involves basolateral Na+, K(+)-ATPase and Na-K-Cl co-transporter. An Na channel has been shown at the apical membrane.

Adenylate cyclase in the semicircular canal : hormonal stimulation and ultrastructural localization

The modulation of the cyclic AMP (cAMP) production and the cytochemical localization of adenylate cyclase were studied in isolated semicircular canal epithelium of the frog. The basal cAMP content, as measured by radioimmunoassay, was 344 +/- 37.8 fmoles/structure/5 min (mean +/- SEM, n = 41). This content was increased 6- to 8-fold by forskolin (10(-7) M to 10(-5) M). Among the tested drugs, only prostaglandin E2, isoproterenol, and vasotocin increased the cAMP production: 1.7-fold by prostaglandin E2 (1.5 X 10(-7) M) and isoproterenol (10(-6) M), and 1.3- and 3.3-fold by vasotocin at 10(-8) M and 10(-7) M, respectively. The addition of alpha 2-adrenergic agonists blunted the stimulatory effect of vasotocin. The adenylate cyclase was evidenced in both the basolateral and apical membranes of the dark cells. Vasotocin stimulated only the apical adenylate cyclase of dark cells. These results indicated that the adenylate cyclase located in the apical dark cells of the semicircular canal was stimulated by the antidiuretic hormone which may be involved in the regulation of the endolymph secretion.