Hanna Hlawaty

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.

Leukotriene Receptor Antagonism and the Prevention of Extracellular Matrix Degradation During Atherosclerosis and In-Stent Stenosis

Objective—The lipid-derived inflammatory mediators leukotrienes (LTs) are produced during vascular injury. The aim of the present study was to determine the role of LT receptor signaling in the pathophysiology of in-stent stenosis. Methods and Results—New Zealand White rabbits were fed 0.3% cholesterol and subjected to angioplasty with balloon dilatation and stent implantation in the right carotid artery. Rabbits treated for 2 weeks with the BLT receptor antagonist BIIL284 (3 mg/kg once daily by oral gavage) displayed a significantly reduced in-stent intimal hyperplasia in carotid arteries compared with vehicle-treated rabbits. In addition, BIIL284 treatment significantly reduced the extracellular matrix metalloproteinase (MMP)-2 and MMP-9 activities in stented arteries. The inhibited MMP-9 activity was correlated with decreased macrophage content in the lesions. The LTB4-induced migration of vascular smooth muscle cells was significantly inhibited by transfection with siRNA against MMP-2. Finally, human arteries subjected to ex vivo angioplasty and stent implantation displayed an increased in-stent intimal hyperplasia and higher MMP-2 and -9 activities in the presence of LTB4. Conclusions—These results suggest a key role of LT signaling in the extracellular matrix degradation associated with hyperlipidemia and in-stent stenosis. In conclusion, targeting LT receptors may represent a therapeutic strategy in atherosclerosis and interventional cardiology.

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.

Development of a Functionalized Polymer for Stent Coating in the Arterial Delivery of Small Interfering RNA

In patients receiving drug eluting stents, there is a growing concern about both the long-term toxicity/degradability of the polymers used for the coating, and the nature of the therapeutic agents. We hypothesized that the use of a functionalized biocompatible polymer for a stent coating could be appropriate for local arterial therapy. A cationized pullulan hydrogel was thus prepared to cover bare metal stents that could be further loaded with small interfering RNA (siRNA) targeted at MMP2 for gene silencing in vascular cells. The efficient coverage of the stent struts by a smooth polymeric layer, which can withstand the crimping of the stent on a balloon-catheter and its deployment, was demonstrated by fluorescence microscopy, scanning electron microscopy, and atomic force microscopy. The release of siRNA from the stents was modulated by the presence of the cationic groups, as compared to noncationized pullulan hydrogel. In vivo implantation of coated stents was successful and cationized pullulan-based hydrogels loaded with siRNA in rabbit balloon-injured carotid arteries induced an uptake of siRNA into the arterial wall and a decrease of pro-MMP2 activity. These results suggest that cationized pullulan-based hydrogel could be used as a new biocompatible and biodegradable stent coating for local gene therapy in the arterial wall.

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.

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.

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.

Comparative toxicity evaluation of flower-shaped and spherical gold nanoparticles on human endothelial cells.

In this paper, we propose a multi-parametric in vitro study of the cytotoxicity of gold nanoparticles (GNPs) on human endothelial cell (HUVEC). The cytotoxicity is evaluated by incubating cells with six different GNP types which have two different morphologies: spherical and flower-shaped, two sizes (∼15 and ∼50 nm diameter) and two surface chemistries (as prepared form and PEGylated form). Our results showed that by increasing the concentration of GNPs the cell viability decreases with a toxic concentration threshold of 10 pM for spherical GNPs and of 1 pM for flower-shaped GNPs. Dark field images, flow cytometry and spreading test revealed that flower-shaped GNPs have more deleterious effects on the cell mechanisms than spherical GNPs. We demonstrated that the main parameter in the evaluation of the GNPs toxicity is the GNPs roughness and that this effect is independent on the surface chemistry. We assume that this behavior is highly related to the efficiency of the GNPs internalization within the cells and that this effect is enhanced due to the specific geometry of the flower-shaped GNPs.

The Oral Cavity and Age: A Site of Chronic Inflammation?

Background Aging may be accompanied by a low grade chronic up-regulation of inflammatory mediators. A variety of endogenous locally released mediators as well as inflammatory cells have been reported in the human oral cavity. The aim of this investigation was to determine the presence of different classes of inflammatory mediators in human saliva and correlate the levels with age. Methodology and Principal Findings Unstimulated whole buccal salivary samples were obtained in the morning from 94 healthy volunteers within 30 minutes after waking. None of the participants had taken aspirin in the week prior to the saliva collection. Lysozyme activity, eicosanoid levels (prostaglandin E2 and leukotriene B4) and MMP-9 activity were measured. The antimicrobial activity (lysozyme activity) was not correlated with age whereas PGE2 levels were markedly correlated with age (r = 0.29; P<0.05; n = 56). Saliva from healthy subjects (≤40 years) compared with data derived from older volunteers (>40 years) demonstrated a significant increase in the mean values for PGE2 and MMP-9 activity with age. In addition, significant correlations were observed between LTB4 and PGE2 (r = 0.28; P<0.05; n = 56) and between LTB4 levels and MMP-9 activity in smokers (r = 0.78; P<0.001; n = 15). Conclusions/Significance The presence of significant levels and activity of inflammatory mediators in saliva suggests that the oral cavity of healthy subjects may be in a constant low state of inflammation associated with age.